WO2026006492A2 - Anticorps anti-prame/hla-a2 et leurs utilisations - Google Patents

Anticorps anti-prame/hla-a2 et leurs utilisations

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Publication number
WO2026006492A2
WO2026006492A2 PCT/US2025/035306 US2025035306W WO2026006492A2 WO 2026006492 A2 WO2026006492 A2 WO 2026006492A2 US 2025035306 W US2025035306 W US 2025035306W WO 2026006492 A2 WO2026006492 A2 WO 2026006492A2
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WO
WIPO (PCT)
Prior art keywords
seq
acid sequence
amino acid
antibody
row
Prior art date
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Pending
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PCT/US2025/035306
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English (en)
Other versions
WO2026006492A3 (fr
Inventor
Dongxing Zha
Jennifer WATKINS-YOON
Jaafar HAIDAR
Melissa LOKUGAMAGE
Lauren Mifflin
William Carson
Pinar AKSOY
Zhiqian Lucy LIU
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ypsilon Therapeutics Inc
Alloy Therapeutics Inc
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Ypsilon Therapeutics Inc
Alloy Therapeutics Inc
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Application filed by Ypsilon Therapeutics Inc, Alloy Therapeutics Inc filed Critical Ypsilon Therapeutics Inc
Publication of WO2026006492A2 publication Critical patent/WO2026006492A2/fr
Publication of WO2026006492A3 publication Critical patent/WO2026006492A3/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2833—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against MHC-molecules, e.g. HLA-molecules
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • PReferentially expressed Antigen in MElanoma is a cancer-associated antigen found to be overexpressed in a variety of cancers, including melanoma, acute myeloid leukemia, non- small cell lung cancer, and other hematological and solid cancers. PRAME is a cancer-testis antigen and under healthy conditions is only expressed in the testis or placenta.
  • the present disclosure relates to the development of antibodies that specifically recognize and bind PRAME peptides when they are complexed with HLA molecules (e.g., HLA-A2).
  • HLA molecules e.g., HLA-A2
  • the disclosure features an isolated antibody (e.g., anti-PRAME/HLA-A2 antibody) that mimics a T cell receptor (TCR) that specifically binds a PRAME/HLA-A2 complex.
  • TCR T cell receptor
  • the present disclosure provides T-cell engaging bispecific antibodies targeting PRAME/HLA-A2, which comprises a first antigen binding site that specifically binds a PRAME/HLA-A2 complex (e.g., any of the antigen binding site derived from any one of the anti-PRAME/HLA-A2 antibody described herein), and a second antigen binding site that specifically binds a T cell antigen (e.g., CD3) (referred to as anti-PRAME/HLA-A2xT cell bispecific antibody).
  • an anti-PRAME/HLA-A2xT cell bispecific antibody recruits a T cell (e.g., cytotoxic T cell) to a cancer cell expressing PRAME.
  • an anti- PRAME/HLA-A2xT cell bispecific antibody elicits T cell mediated immune response (e.g., cytotoxic T cell mediated immune response) against a cancer cell expressing PRAME.
  • T cell mediated immune response e.g., cytotoxic T cell mediated immune response
  • an anti-PRAME/HLA-A2xT cell bispecific antibody induces T cell mediated killing (e.g., cytotoxic T cell mediated immune response) of a cancer cell expressing PRAME.
  • an anti-PRAME/HLA-A2xT cell bispecific antibody is useful for treating cancer (e.g., cancer expressing PRAME).
  • the present disclosure also provides a chimeric T cell receptor (CAR) that comprises an antigen binding site of an anti-PRAME/HLA-A2.
  • CAR chimeric T cell receptor
  • a chimeric T cell expressing a CAR comprising an antigen binding site of an anti- PRAME/HLA-A2 can be used for treating cancer (e.g., cancer expressing PRAME).
  • the present disclosure provides a means for specifically binding a peptide/HLA-A2 complex, wherein the peptide/HLA-A2 complex is a member of a set of complexes of peptides bound by HLA-A2, each complex comprising a different peptide bound by HLA-A2, wherein each peptide has an amino acid sequence defined by a formula set forth as X56X57X58X59HLIGX60 (SEQ ID NO: 18).
  • the present disclosure provides a means for specifically binding a PRAME/HLA-A2 complex, wherein the PRAME/HLA-A2 complex comprises a PRAME peptide comprising the amino acid sequence of SLLQHLIGL (SEQ ID NO: 19) bound by HLA-A2.
  • the means for specifically binding a peptide/HLA-A2 complex e.g., peptide X56X57X58X59HLIGX60 (SEQ ID NO: 18) bound by HLA-A2
  • the means for specifically binding a PRAME/HLA-A2 complex e.g., PRAME peptide SLLQHLIGL (SEQ ID NO: 19) bound by HLA-A2
  • a PRAME/HLA-A2 complex e.g., PRAME peptide SLLQHLIGL (SEQ ID NO: 19) bound by HLA-A2
  • an anti- PRAME/HLA-A2 antibody described herein e.g., an antibody described in Table 1 or equivalents thereof.
  • the present disclosure provides a means for specifically binding a peptide/HLA-A2 complex, wherein the peptide/HLA-A2 complex is a member of a set of complexes of peptides bound by HLA-A2, each complex comprising a different peptide bound by HLA-A2, wherein each peptide has an amino acid sequence defined by a formula set forth as X53X54X55X56X57LIGX58.
  • the present disclosure provides a means for specifically binding a PRAME/HLA-A2 complex, wherein the PRAME/HLA-A2 complex comprises a PRAME peptide comprising the amino acid sequence of SLLQHLIGL (SEQ ID NO: 19) bound by HLA-A2.
  • the means for specifically binding a peptide/HLA-A2 complex e.g., peptide X53X54X55X56X57LIGX58 bound by HLA- A2
  • the means for specifically binding a PRAME/HLA-A2 complex e.g., PRAME peptide SLLQHLIGL (SEQ ID NO: 19) bound by HLA-A2
  • a PRAME/HLA-A2 complex e.g., PRAME peptide SLLQHLIGL (SEQ ID NO: 19) bound by HLA-A2
  • an anti-PRAME/HLA-A2 antibody described herein e.g., an antibody described in Table 1 or equivalents thereof.
  • the present disclosure provides means for engaging a T cell to a cancer cell expressing PRAME.
  • the means for engaging a T cell to a cancer cell expressing PRAME is an anti-PRAME/HLA-A2xT cell bispecific antibody described herein.
  • the present disclosure provides means for eliciting cytotoxic T cell immunity against cancer cells expressing PRAME.
  • the means for eliciting cytotoxic T cell immunity against cancer cells expressing PRAME is an anti-PRAME/HLA-A2xT cell bispecific antibody described herein.
  • the present disclosure provides an antibody that specifically binds PRAME/HLA-A2 complex, wherein the improvement comprises one or more of the HC CDRs (e.g., HC CDR1, HC CDR2, or HC CDR3) amino acid sequences from any one of the anti-PRAME/HLA-A2 antibodies selected from Table 1.
  • the present disclosure provides an antibody that specifically binds PRAME/HLA-A2 complex, wherein the improvement comprises the HC CDR1, HC CDR2, and HC CDR3 as provided for any one of the antibodies selected from Table 1.
  • the present disclosure provides an antibody that specifically binds PRAME/HLA-A2 complex, wherein the improvement comprises one or more of the LC CDRs (e.g., LC CDR1, LC CDR2, or LC CDR3) amino acid sequences from any one of the anti-PRAME/HLA-A2 antibodies selected from Table 1.
  • the present disclosure provides an antibody that specifically binds PRAME/HLA-A2 complex, wherein the improvement comprises the LC CDR1, LC CDR2, and LC CDR3 as provided for any one of the anti-PRAME/HLA-A2 antibodies selected from Table 1.
  • the present disclosure provides an antibody that specifically binds PRAME/HLA-A2 complex, wherein the improvement comprises HC CDR1, HC CDR2, and HC CDR3, LC CDR1, LC CDR2, and LC CDR3 as provided for any one of the anti-PRAME/HLA-A2 antibodies selected from Table 1.
  • the present disclosure provides means for engaging a T cell to a cancer cell expressing PRAME.
  • the means for engaging a T cell to a cancer cell expressing PRAME is an anti-PRAME/HLA-A2xT cell bispecific antibody described herein.
  • the present disclosure provides means for eliciting cytotoxic T cell immunity against cancer cells expressing PRAME.
  • the means for eliciting cytotoxic T cell immunity against cancer cells expressing PRAME is an anti-PRAME/HLA-A2xT cell bispecific antibody described herein.
  • the present disclosure provides an antibody that specifically binds PRAME/HLA-A2 complex, wherein the improvement comprises one or more of the HC CDRs (e.g., HC CDR1, HC CDR2, or HC CDR3) amino acid sequences from any one of the anti-PRAME/HLA-A2 antibodies selected from Table 1.
  • the present disclosure provides an antibody that specifically binds PRAME/HLA-A2 complex, wherein the improvement comprises the HC CDR1, HC CDR2, and HC CDR3 as provided for any one of the antibodies elected from Table 1.
  • the present disclosure provides an antibody that specifically binds PRAME/HLA-A2 complex, wherein the improvement comprises one or more of the LC CDRs (e.g., LC CDR1, LC CDR2, or LC CDR3) amino acid sequences from any one of the anti-PRAME/HLA-A2 antibodies selected from Table 1.
  • the present disclosure provides an antibody that specifically binds PRAME/HLA-A2 complex, wherein the improvement comprises the LC CDR1, LC CDR2, and LC CDR3 as provided for any one of the anti-PRAME/HLA-A2 antibodies selected from Table 1.
  • the present disclosure provides an antibody that specifically binds PRAME/HLA-A2 complex, wherein the improvement comprises HC CDR1, HC CDR2, and HC CDR3, LC CDR1, LC CDR2, and LC CDR3 as provided for any one of the anti-PRAME/HLA-A2 antibodies selected from Table 1.
  • the present disclosure provides means for engaging a T cell to a cancer cell expressing PRAME.
  • the means for engaging a T cell to a cancer cell expressing PRAME is an anti-PRAME/HLA-A2xT cell bispecific antibody described herein.
  • the present disclosure provides means for eliciting cytotoxic T cell immunity against cancer cells expressing PRAME.
  • the means for eliciting cytotoxic T cell immunity against cancer cells expressing PRAME is an anti-PRAME/HLA-A2xT cell bispecific antibody described herein.
  • the present disclosure provides an antibody that specifically binds PRAME/HLA-A2 complex, wherein the improvement comprises one or more of the HC CDRs (e.g., HC CDR1, HC CDR2, or HC CDR3) amino acid sequences from any one of the anti-PRAME/HLA-A2 antibodies selected from Table 1.
  • the present disclosure provides an antibody that specifically binds PRAME/HLA-A2 complex, wherein the improvement comprises the HC CDR1, HC CDR2, and HC CDR3 as provided for any one of the antibodies elected from Table 1.
  • the present disclosure provides an antibody that specifically binds PRAME/HLA-A2 complex, wherein the improvement comprises one or more of the LC CDRs (e.g., LC CDR1, LC CDR2, or LC CDR3) amino acid sequences from any one of the anti-PRAME/HLA-A2 antibodies selected from Table 1.
  • the present disclosure provides an antibody that specifically binds PRAME/HLA-A2 complex, wherein the improvement comprises the LC CDR1, LC CDR2, and LC CDR3 as provided for any one of the anti-PRAME/HLA-A2 antibodies selected from Table 1.
  • the present disclosure provides an antibody that specifically binds PRAME/HLA-A2 complex, wherein the improvement comprises HC CDR1, HC CDR2, and HC CDR3, LC CDR1, LC CDR2, and LC CDR3 as provided for any one of the anti-PRAME/HLA-A2 antibodies selected from Table 1.
  • the present disclosure provides means for engaging a T cell to a cancer cell expressing PRAME.
  • the means for engaging a T cell to a cancer cell expressing PRAME is an anti-PRAME/HLA-A2xT cell bispecific antibody described herein.
  • the present disclosure provides means for eliciting cytotoxic T cell immunity against cancer cells expressing PRAME.
  • the means for eliciting cytotoxic T cell immunity against cancer cells expressing PRAME is an anti-PRAME/HLA-A2xT cell bispecific antibody described herein.
  • the present disclosure provides an antibody that specifically binds PRAME/HLA-A2 complex, wherein the improvement comprises one or more of the HC CDRs (e.g., HC CDR1, HC CDR2, or HC CDR3) amino acid sequences from any one of the anti-PRAME/HLA-A2 antibodies selected from Table 1.
  • the present disclosure provides an antibody that specifically binds PRAME/HLA-A2 complex, wherein the improvement comprises the HC CDR1, HC CDR2, and HC CDR3 as provided for any one of the antibodies elected from Table 1.
  • the present disclosure provides an antibody that specifically binds PRAME/HLA-A2 complex, wherein the improvement comprises one or more of the LC CDRs (e.g., LC CDR1, LC CDR2, or LC CDR3) amino acid sequences from any one of the anti-PRAME/HLA-A2 antibodies selected from Table 1.
  • the present disclosure provides an antibody that specifically binds PRAME/HLA-A2 complex, wherein the improvement comprises the LC CDR1, LC CDR2, and LC CDR3 as provided for any one of the anti-PRAME/HLA-A2 antibodies selected from Table 1.
  • the present disclosure provides an antibody that specifically binds PRAME/HLA-A2 complex, wherein the improvement comprises HC CDR1, HC CDR2, and HC CDR3, LC CDR1, LC CDR2, and LC CDR3 as provided for any one of the anti-PRAME/HLA-A2 antibodies selected from Table 1.
  • the present disclosure provides means for engaging a T cell to a cancer cell expressing PRAME.
  • the means for engaging a T cell to a cancer cell expressing PRAME is an anti-PRAME/HLA-A2xT cell bispecific antibody described herein.
  • the present disclosure provides means for eliciting cytotoxic T cell immunity against cancer cells expressing PRAME.
  • the means for eliciting cytotoxic T cell immunity against cancer cells expressing PRAME is an anti-PRAME/HLA-A2xT cell bispecific antibody described herein.
  • the present disclosure provides an antibody that specifically binds PRAME/HLA-A2 complex, wherein the improvement comprises one or more of the HC CDRs (e.g., HC CDR1, HC CDR2, or HC CDR3) amino acid sequences from any one of the anti-PRAME/HLA-A2 antibodies selected from Table 1.
  • the present disclosure provides an antibody that specifically binds PRAME/HLA-A2 complex, wherein the improvement comprises the HC CDR1, HC CDR2, and HC CDR3 as provided for any one of the antibodies elected from Table 1.
  • the present disclosure provides an antibody that specifically binds PRAME/HLA-A2 complex, wherein the improvement comprises one or more of the LC CDRs (e.g., LC CDR1, LC CDR2, or LC CDR3) amino acid sequences from any one of the anti-PRAME/HLA-A2 antibodies selected from Table 1.
  • the present disclosure provides an antibody that specifically binds PRAME/HLA-A2 complex, wherein the improvement comprises the LC CDR1, LC CDR2, and LC CDR3 as provided for any one of the anti-PRAME/HLA-A2 antibodies selected from Table 1.
  • the present disclosure provides an antibody that specifically binds PRAME/HLA-A2 complex, wherein the improvement comprises HC CDR1, HC CDR2, and HC CDR3, LC CDR1, LC CDR2, and LC CDR3 as provided for any one of the anti-PRAME/HLA-A2 antibodies selected from Table 1.
  • an anti-PRAME/HLA-A2xT cell bispecific antibody comprises a second antigen binding site that specifically binds to CD3, wherein the improvement comprises a first antigen binding site that specifically binds to a PRAME/HLA- A2 complex that comprises any one of the anti-PRAME/HLA-A2 binding sites derived from any of the anti-PRAME/HLA-A2 antibodies described herein (e.g., any one of the anti- PRAME/HLA-A2 binders described in Table 1).
  • the disclosure provides an antibody that specifically binds PRAME (PReferentially expressed Antigen in MElanoma)/HLA-A2 complex (PRAME/HLA-A2) comprising (a) a heavy chain complementarity determining region 1 (HC CDR1), a heavy chain complementarity determining region 2 (HC CDR2), and a heavy chain complementarity determining region 3 (HC CDR3) of a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 27, and a light chain complementarity determining region 1 (LC CDR1), a light chain complementarity determining region 2 (LC CDR2), and a light chain complementarity determining region 3 (LC CDR3) of a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 28; (b) a HC CDR1, a HC CDR2, a HC CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 31, and a LC
  • an antibody comprises (a) a HC CDR1 comprising the amino acid sequence of SEQ ID NO: 21, a HC CDR2 comprising the amino acid sequence of SEQ ID NO: 22, a HC CDR3 comprising the amino acid sequence of SEQ ID NO: 23, a LC CDR1 comprising the amino acid sequence of SEQ ID NO: 24, a LC CDR2 comprising the amino acid sequence of SEQ ID NO: 25, and a LC CDR3 comprising the amino acid sequence of SEQ ID NO: 26; (b) a HC CDR1 comprising the amino acid sequence of SEQ ID NO: 29, a HC CDR2 comprising the amino acid sequence of SEQ ID NO: 22, a HC CDR3 comprising the amino acid sequence of SEQ ID NO: 30, a LC CDR1 comprising the amino acid sequence of SEQ ID NO: 24, a LC CDR2 comprising the amino acid sequence of SEQ ID NO: 25, and a LC CDR3 comprising the amino acid sequence of
  • an antibody comprises (a) a VH comprising the amino acid of SEQ ID NO: 27, and/or a VL comprising the amino acid sequence of SEQ ID NO: 28; (b) a VH comprising the amino acid of SEQ ID NO: 31, and/or a VL comprising the amino acid sequence of SEQ ID NO: 28; (c) a VH comprising the amino acid of SEQ ID NO: 38, and/or a VL comprising the amino acid sequence of SEQ ID NO: 32; (d) a VH comprising the amino acid of SEQ ID NO: 42, and/or a VL comprising the amino acid sequence of SEQ ID NO: 32; (e) a VH comprising the amino acid of SEQ ID NO: 39, and/or a VL comprising the amino acid sequence of SEQ ID NO: 32; (f) a VH comprising the amino acid sequence of SEQ ID NO: 87, and/or a VL comprising the amino acid sequence of SEQ ID NO:
  • an antibody is a full-length IgG, a Fab fragment, a F(ab') fragment, a F(ab’)2 fragment, a scFv, or a Fv.
  • an antibody comprises a heavy chain constant region of the isotype IgGl, IgG2, IgG3, or IgG4.
  • the disclosure provides an antibody that specifically binds members of a set of complexes of peptides bound by HLA-A2, each complex comprising a different peptide bound by HLA-A2, wherein each peptide has an amino acid sequence defined by a formula set forth as X56X57X58X59HLIGX60 (SEQ ID NO: 18), wherein X56 is S,
  • At least one member of the set of complexes comprises a PRAME peptide.
  • the PRAME peptide comprises the amino acid sequence consisting of SLLQHLIGL (SEQ ID NO: 19).
  • the disclosure provides an antibody that specifically binds members of a set of complexes of peptides bound by HLA-A2, each complex comprising a different peptide bound by HLA-A2, wherein each peptide has an amino acid sequence defined by a formula set forth as X53X54X55X56X57LIGXs 8 , wherein X53 is S, G, A, T, Y, Q,
  • X54 is M or L when X53 is S, X55 is L, X56 is Q, X57 is H, and Xs 8 is L;
  • X54 is M or L when X53 is S, X55 is L, X56 is Q, X57 is H, and Xs 8 is L;
  • X55 is Y, M, L, F, H, D, or W when X53 is S, X54 is L, X56 is Q, X57 is H, and Xs 8 is L;
  • X56 is D, E, P, N, G, S, Q, T, K, or A when X53 is S, X54 is E, X55 is E, X57 is H, and X 58 is L;
  • X57 is W, S, A, N, H, G, T, or S when X53 is S, X54 is E, X55 is L, X56 is Q, and X58 is L; or Xss
  • an antibody is obtained by a process comprising (1) exposing members of the set of complexes to a library of antibodies under conditions in which at least one antibody of the library that specifically binds to the members of the set of complexes is detected; (2) obtaining the sequence of a heavy chain variable domain of an antibody detected in step (1) that specifically binds the members of the set of complexes; and (3) producing an antibody having at least a heavy chain complementarity determining region 3 (HC CDR3) of the heavy chain variable domain of step (2), thereby obtaining the antibody that specifically binds to the members of the set of complexes.
  • an antibody is obtained by a process that further comprises (4) confirming that the antibody obtained by step (3) binds a PRAME peptide bound by HLA-A2.
  • the disclosure provides a bispecific antibody comprising a first antigen binding site comprising an antibody provided herein, and a second antigen binding site.
  • the second antigen binding site specifically binds to a T cell antigen.
  • the T cell antigen is a CD3 complex.
  • the second antigen binding site specifically binds CD35 of a CD3 complex.
  • the second antigen binding site specifically binds CD3s of a CD3 complex.
  • the second antigen binding site specifically binds CD3y of a CD3 complex.
  • the second antigen binding site specifically binds a CD3s/5 heterodimer of a CD3 complex.
  • the second antigen binding site specifically binds a CD3s/y heterodimer of a CD3 complex.
  • the bispecific antibody comprises a first arm that is configured as a Fab, a Fab’, or a scFv and that comprises the first antigen binding site. In some embodiments, the bispecific antibody comprises a second arm that is configured as a Fab, a Fab’, or a scFv and that comprises the second antigen binding site. In some embodiments, the bispecific antibody comprises a first arm that is configured as a Fab and a second arm that is configured as a scFv. In some embodiments, the bispecific antibody comprises a first arm that is configured as a scFv and a second arm that is configured as a Fab.
  • the ratio between the first antigen binding site and the second antigen binding site is 1:1, 1:2, 1:3, 2:1 or 3:1.
  • the disclosure provides a chimeric antigen receptor (CAR) comprising an antibody provided herein.
  • the disclosure provides an isolated nucleic acid encoding the VH and/or VL of an antibody provided herein, a bispecific antibody, or a CAR provided herein.
  • the isolated nucleic acid comprises (a) the nucleic acid sequence of SEQ ID NO: 43, and/or the nucleic acid sequence of SEQ ID NO: 44; (b) the nucleic acid sequence of SEQ ID NO: 45, and/or the nucleic acid sequence of SEQ ID NO: 44; (c) the nucleic acid sequence of SEQ ID NO: 46, and/or the nucleic acid sequence of SEQ ID NO: 47; (d) the nucleic acid sequence of SEQ ID NO: 16, and/or the nucleic acid sequence of SEQ ID NO: 47; or (e) the nucleic acid sequence of SEQ ID NO: 48, and/or the nucleic acid sequence of SEQ ID NO: 47.
  • the disclosure provides an isolated nucleic acid encoding the VH and/or VL of an antibody provided herein, a bispecific antibody provided herein, or a CAR provided herein.
  • the isolated nucleic acid comprises (a) the nucleic acid sequence of SEQ ID NO: 43, and/or the nucleic acid sequence of SEQ ID NO: 44; (b) the nucleic acid sequence of SEQ ID NO: 45, and/or the nucleic acid sequence of SEQ ID NO: 57; (c) the nucleic acid sequence of SEQ ID NO: 46, and/or the nucleic acid sequence of SEQ ID NO: 47; (d) the nucleic acid sequence of SEQ ID NO: 48, and/or the nucleic acid sequence of SEQ ID NO: 58; (e) the nucleic acid sequence of SEQ ID NO: 49, and/or the nucleic acid sequence of SEQ ID NO: 59; or (f) the nucleic acid sequence of SEQ ID NO:
  • the disclosure provides an expression vector comprising an isolated nucleic acid provided herein.
  • the disclosure provides a host cell comprising an antibody, a bispecific antibody, a CAR, an isolated nucleic acid, or an expression vector provided herein.
  • the disclosure provides an engineered cell expressing an antibody, a bispecific antibody, or a CAR provided herein.
  • the engineered cell is a T cell, a NK cell, or a NKT cell.
  • the disclosure provides a composition comprising an antibody, a bispecific antibody, a CAR, an isolated nucleic acid, an expression vector, a host cell, or an engineered cell provided herein.
  • the composition further comprises a pharmaceutically acceptable carrier.
  • the disclosure provides a method of treating cancer, the method comprising administering to a subject in need thereof an effective amount of an antibody, a bispecific antibody, a CAR, an isolated nucleic acid, an expression vector, a host cell, an engineered cell, or a composition provided herein.
  • FIG. 1 is a high-level schematic of the lead antibody selection process.
  • FIGs. 2A-2B are schema of the phage -based poly specificity assay. Phages were loaded with PRAME peptides complexed with single-chain HLA-A2 molecules (FIG. 2A). 100 PRAME peptides were complexed with single-chain HLA-A2 molecules and loaded onto phages for high-throughput screens (FIG. 2B).
  • FIG. 3 shows binding strength of candidate antibodies against two PRAME peptides - VLD (full peptide sequence VLDGLDVLL (SEQ ID NO: 17)) and SLL (full peptide sequence SLLQHLIGL (SEQ ID NO: 19)) - as well as binding to off-target peptide/HLA-A2 complexes.
  • FIG. 4 are kinetics sensorgrams for each lead binder showing the relative response (RU) to a PRAME peptide.
  • FIG. 5 is a workflow schematic for epitope scanning of residues important for lead antibody recognition of a peptide, e.g., a PRAME peptide, complexed to a single-chain HLA- A2 molecule.
  • a peptide e.g., a PRAME peptide
  • Each residue of the peptide was individually and sequentially substituted for every possible amino acid.
  • Each iteration was complexed with HLA-A2 and loaded onto a phage for monovalent presentation to lead antibodies, after which next-generation sequencing was performed to determine the residues of the peptides to which the lead antibodies bind. Sequences shown correspond (top-bottom) to SEQ ID NOs: 49-58.
  • FIG. 6 shows the binding strength of control beads (left) and of an example antibody lead (right) to each iteration of the SLL PRAME peptide.
  • each row represents an amino acid residue
  • each column represents an amino acid position within the SLL peptide. Darker squares in the heat map represent strong binding to the peptide for a given residue substitution.
  • Below the heatmap is an amino acid sequence logo plot. The relative size of the depiction of the residue corresponds to its frequency in the iterations of the peptides that are bound by the control beads of antibody candidates. Sequences shown correspond (top-bottom) to SEQ ID NOs: 59-68.
  • FIG. 7 is an amino acid sequence logo plot for an antibody described herein that binds the SLL PRAME peptide. Sequence shown corresponds to SEQ ID NO: 19.
  • PRAME PReferentially expressed Antigen in MElanoma
  • PRAME is one of the most highly expressed cancer-associated antigens. Under homeostatic conditions, PRAME is expressed intracellularly only in the testis or placenta and is thus categorized as a cancer-testis antigen. The normal function of PRAME is not fully understood, but it is thought to promote cancer cell proliferation, survival, and immune evasion. While PRAME is normally only expressed intracellularly, in cancer cells it is broken down into peptides and peptide fragments, which are then bound by HLA molecules for display on the cancer cell surface.
  • PRAME PReferentially expressed Antigen in MElanoma
  • PRAME expression is a protein found to be expressed by cells in a number of cancers, including melanoma, leukemia, breast cancer, lung cancer, and others. In healthy conditions, PRAME expression is limited to germline cells (e.g., sperm cells) and is not expressed in somatic cells.
  • PRAME is an intracellular protein thought to function as a repressor of retinoic acid signaling, but in cancer cells it is processed by the proteosome such that PRAME peptides are presented on MHC or HLA molecules on the cancer cell surface, forming a PRAME/MHC complex (e.g., a PRAME/MHC-I complex) or a PRAME/HLA complex (e.g., a PRAME/HLA-A2 complex).
  • PRAME/MHC complex e.g., a PRAME/MHC-I complex
  • PRAME/HLA complex e.g., a PRAME/HLA-A2 complex.
  • Such complexes can be recognized by immune cells, including T effector cells.
  • HLA molecules recognized by CD4+ T cells are encoded by the HLA-DP, HLA-DQ, and HLA-DR genes, while HLA molecules recognized by CD8+ T cells are encoded by the HLA- A, HLA-B, and HLA-C genes.
  • Different alleles of genes encoding HLA molecules result in different HLA serotypes, such as HLA-A2 in the case of HLA-A.
  • an antibody provided herein specifically binds PRAME complexed with HLA- A2 (also referred to as HLA-A2, HLA-A02, HLA-A*02, or HLA-A*2).
  • HLA-A2 can be further serotyped and separated by allele variation (e.g., HLA-A2:01, HLA-A2:01, HLA- A2:02, HLA-A2:03, HLA-A2:04, HLA-A2:05, HLA-A2:06, HLA-A2:07, HLA-A2:11, etc.).
  • an anti-PRAME/HLA-A2 antibody specifically binds PRAME complexed with HLA-A serotype HLA-A*02:01.
  • an anti-PRAME/HLA-A2 is a bispecific antibody and comprises a first binding site that specifically binds PRAME/HLA-A2 and a second binding site.
  • the second binding site specifically binds a T cell antigen.
  • the bispecific antibody can act as a scaffold, bringing a T cell to a PRAME/HLA-A2 expressing cell, e.g., a PRAME/HLA-A2 expressing cancer cell, to induce an immune response against the PRAME/HLA-A2 expressing cell.
  • an anti- PRAME/HLA-A2 antibody and/or an immune cell comprising a chimeric antigen receptor (CAR) comprising an anti-PRAME/HLA-A2 antibody is used to treat cancer in a subject.
  • the antibodies described herein show high binding affinity and specificity for PRAME/HLA-A2. The generation of the highly specific anti-PRAME/HLA- A2 antibodies provided by the present disclosure represents a significant advancement in the field.
  • Administering means to provide an antibody or a composition thereof to a subject in a manner that is physiologically and/or pharmacologically useful (e.g., to treat a condition in the subject).
  • Affinity Matured Antibody “Affinity Matured Antibody” is used herein to refer to an antibody with one or more alterations in one or more CDRs, which result in an improvement in the affinity (i.e. KD, kd or ka) of the antibody for a target antigen compared to a parent antibody, which does not possess the alteration(s).
  • Exemplary affinity matured antibodies will have nanomolar or even picomolar affinities for the target antigen.
  • an antibody refers to a polypeptide that includes at least one immunoglobulin variable domain or at least one site, e.g., paratope, that specifically binds to an antigen.
  • an antibody comprises a paratope.
  • a paratope comprises one or more complementarity determining regions (CDRs).
  • CDRs complementarity determining regions
  • an antibody is a full-length antibody.
  • an antibody is a chimeric antibody.
  • an antibody is a humanized antibody.
  • an antibody is a Fab fragment, a Fab’ fragment, a F(ab')2 fragment, a Fv fragment or a scFv fragment.
  • an antibody is a nanobody derived from a camelid antibody or a nanobody derived from shark antibody.
  • an antibody is a diabody.
  • an antibody comprises a framework having a human germline sequence.
  • an antibody comprises a heavy chain constant domain selected from the group consisting of IgG, IgGl, IgG2, IgG2A, IgG2B, IgG2C, IgG3, IgG4, IgAl, IgA2, IgD, IgM, and IgE constant domains.
  • an antibody comprises a heavy (H) chain variable region (abbreviated herein as VH), and/or a light (L) chain variable region (abbreviated herein as VL).
  • an antibody comprises a constant domain, e.g., an Fc region.
  • An immunoglobulin constant domain refers to a heavy or light chain constant domain.
  • the heavy chain of an antibody described herein can be an alpha (a), delta (A), epsilon (e), gamma (y) or mu (p) heavy chain.
  • the heavy chain of an antibody described herein can comprise a human alpha (a), delta (A), epsilon (e), gamma (y) or mu (p) heavy chain.
  • an antibody described herein comprises a human gamma 1 CHI, CH2, and/or CH3 domain.
  • the amino acid sequence of the VH domain comprises the amino acid sequence of a human gamma (y) heavy chain constant region, such as any known in the art.
  • a human constant region sequence such as any known in the art.
  • human constant region sequences have been described in the art, e.g., see U.S. Pat. No. 5,693,780 and Kabat E A et al., (1991) supra.
  • the VH domain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or at least 99% identical to any of the variable chain constant regions provided herein.
  • an antibody is modified, e.g., modified via glycosylation, phosphorylation, sumoylation, and/or methylation.
  • an antibody is a glycosylated antibody, which is conjugated to one or more sugar or carbohydrate molecules.
  • the one or more sugar or carbohydrate molecules are conjugated to an antibody via N-glycosylation, O-glycosylation, C-glycosylation, glypiation (GPI anchor attachment), and/or phosphoglycosylation.
  • the one or more sugar or carbohydrate molecules are monosaccharides, disaccharides, oligosaccharides, or glycans.
  • the one or more sugar or carbohydrate molecule is a branched oligosaccharide or a branched glycan.
  • the one or more sugar or carbohydrate molecule includes a mannose unit, a glucose unit, an N-acetylglucosamine unit, or a phospholipid unit.
  • an antibody is a construct that comprises a polypeptide comprising one or more antigen binding fragments of the disclosure linked to a linker polypeptide or an immunoglobulin constant domain.
  • Linker polypeptides comprise two or more amino acid residues joined by peptide bonds and are used to link one or more antigen binding portions. Examples of linker polypeptides have been reported (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci.
  • an antibody may be part of a larger immunoadhesion molecule, formed by covalent or noncovalent association of the antibody or antibody portion with one or more other proteins or peptides.
  • immunoadhesion molecules include use of the streptavidin core region to make a tetrameric scFv molecule (Kipriyanov, S. M., et al.
  • an antibody can be a bispecific and/or a multispecific antibody.
  • Bispecific Antibody refers to a polypeptide or a complex (e.g., two covalently linked polypeptides) that includes two different antigen binding sites, e.g., paratopes, that have different antigen binding specificities.
  • a bispecific antibody is a polypeptide that includes two different antigen binding sites in which each antigen binding site binds to a different epitope of the same antigen.
  • a bispecific antibody is a polypeptide that includes two different antigen binding sites in which each site binds to a different antigen.
  • a bispecific antibody comprises two different sets of immunoglobulin variable domains, each of which set binds to a different epitope or group of epitopes.
  • at least one antigen to which an antigen binding site of a bispecific antibody specifically binds is a complex (e.g., a peptide complexed with a single chain HLA-A2 molecule).
  • each of the two antigen binding sites is present within an “arm” of a bispecific antibody.
  • an arm of a bispecific antibody is configured as a monospecific antibody, e.g., a full-length IgG or a fragment thereof.
  • a bispecific antibody comprises two arms, one or each of which is configured as an Fv region that confers specificity to distinct antigen residues.
  • a bispecific antibody comprises two arms of the same configuration, e.g., a Fab, a Fab’, a scFv, or any other suitable format.
  • a bispecific antibody comprises two arms of two different configurations, e.g., a Fab on one arm and a scFv on the other arm. In some embodiments, a bispecific antibody does not comprise an Fc region. In some embodiments, the two arms of a bispecific antibody are linked directly. In some embodiments, the two arms of a bispecific antibody are linked by a linker. In some embodiments, a bispecific antibody comprises an Fc region, e.g., a dimeric Fc or a monomeric Fc. In some embodiments, a bispecific antibody comprises a monomeric Fc.
  • a bispecific antibody comprises one arm linked to one end (e.g., the N terminal) of a monomeric Fc and a second arm linked to the other end (e.g., the C terminal) of the monomeric Fc (see, e.g., Shan et al., “In vivo pharmacokinetic enhancement of monomeric Fc and monovalent bispecific designs through structural guidance”, Communications Biology, Vol. 4, Article No. 1048 (2021)).
  • a bispecific antibody comprises a dimeric Fc region.
  • a bispecific antibody comprises two arms that are oriented symmetrically around an Fc region (e.g., each Fc monomer of a dimeric Fc region is linked to one arm).
  • a bispecific antibody comprises two distinct heavy chains and two distinct light chains, with each heavy chain/light chain pair having different antigen binding specificity.
  • a bispecific antibody comprises two arms that are oriented asymmetrically around an Fc (e.g., the two arms of the bispecific antibody are linked to one of the monomers of a dimeric Fc region).
  • bispecific antibodies because bispecific antibodies are capable of binding two different targets, they can be used as scaffolds to recruit or redirect cells to antigenic targets, e.g., to recruit immune cells to cancer cells.
  • a bispecific antibody contains a first antigen binding site that specifically binds a peptide:MHC complex (e.g., PRAME/HLA-A2 complex) and a second antigen binding site.
  • a bispecific antibody comprises a second antigen binding site specifically binds to a T cell antigen.
  • the T cell antigen is a CD3 complex or a portion thereof, e.g., CD38, CD3e, CD3y, a CD3e/8 heterodimer, or a CD3e/y heterodimer.
  • a bispecific antibody comprises a first antigen binding site structured as a T cell receptor-mimic (TCRm) antibody that specifically binds to a peptide:MHC complex (e.g., PRAME/HLA-A2 complex), and a second binding site that specifically binds to a T cell antigen (e.g., CD3).
  • TCRm T cell receptor-mimic
  • CDR refers to the complementarity determining region within antibody variable sequences.
  • a typical antibody molecule comprises a heavy chain variable region (VH) and a light chain variable region (VL), which are usually involved in antigen binding.
  • VH and VL regions can be further subdivided into regions of hypervariability, also known as “complementarity determining regions” (“CDR”), interspersed with regions that are more conserved, which are known as “framework regions” (“FR”).
  • CDR complementarity determining regions
  • FR framework regions
  • Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the extent of the framework region and CDRs can be precisely identified using methodology known in the art, for example, by the Kabat definition, the IMGT definition, the Chothia definition, the AbM definition, and/or the contact definition, all of which are well known in the art. See, e.g., Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242; IMGT®, the international ImMunoGeneTics information system® http://www.imgt.org, Lefranc, M.-P.
  • a CDR may refer to the CDR defined by any method known in the art. Two antibodies having the same CDR means that the two antibodies have the same amino acid sequence of that CDR as determined by the same method, for example, the IMGT definition.
  • CDR set refers to a group of three CDRs that occur in a single variable region capable of binding the antigen.
  • the exact boundaries of these CDRs have been defined differently according to different systems.
  • the system described by Kabat Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides an unambiguous residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries defining the three CDRs.
  • CDRs may be referred to as Kabat CDRs.
  • Sub-portions of CDRs may be designated as LC CDR1, LC CDR2 and LC CDR3 or HC CDR1, HC CDR2 and HC CDR3 where the "LC” and the "HC” designate the light chain and the heavy chains regions, respectively.
  • These regions may be referred to as Chothia CDRs, which have boundaries that overlap with Kabat CDRs.
  • Other boundaries defining CDRs overlapping with the Kabat CDRs have been described by Padlan (FASEB J. 9:133- 139 (1995)) and MacCallum (J Mol Biol 262(5) :732-45 (1996)).
  • CDR boundary definitions may not strictly follow one of the above systems, but will nonetheless overlap with the Kabat CDRs, although they may be shortened or lengthened in light of prediction or experimental findings that particular residues or groups of residues or even entire CDRs do not significantly impact antigen binding.
  • the methods used herein may utilize CDRs defined according to any of these systems, although preferred embodiments use Kabat or Chothia defined CDRs.
  • the CDRs of an antibody may have different amino acid sequences when different definition systems are used (e.g., the IMGT definition, the Kabat definition, or the Chothia definition).
  • a definition system annotates each amino acid in a given antibody sequence (e.g., VH or VL sequence) with a number, and numbers corresponding to the heavy chain and light chain CDRs are provided in Table 2.
  • the CDRs listed in Table 1 are defined in accordance with the Kabat definition.
  • One skilled in the art is able to derive the CDR sequences using the different numbering systems for the anti- PRAME/HLA-A2 antibodies provided in Table 1.
  • CDR-grafted antibody refers to antibodies which comprise heavy and light chain variable region sequences from one species but in which the sequences of one or more of the CDR regions of VH and/or VL are replaced with CDR sequences of another species, such as antibodies having murine heavy and light chain variable regions in which one or more of the murine CDRs (e.g., CDR3) has been replaced with human CDR sequences.
  • CDR-grafted antibody refers to antibodies which comprise heavy and light chain variable region sequences from one species but in which the sequences of one or more of the CDR regions of VH and/or VL are replaced with CDR sequences of another species, such as antibodies having murine heavy and light chain variable regions in which one or more of the murine CDRs (e.g., CDR3) has been replaced with human CDR sequences.
  • chemotherapeutic agent refers to a chemical compound useful in the treatment of proliferative disorders, such as cancers (e.g., cancers expressing CD22).
  • these agents can be, e.g., alkylating agents, such as thiotepa and cyclophosphamide (CYTOXAN®); alkylsulfonates such as busulfan, improsulfan and piposulfane; aziridines such as benzodopa, carbocuone, meturedopa and uredopa; ethylene imines and methylamelamines, including altretamine, triethylene methamine, triethylene phosphoramide, triethylene-thiophosphoramide and trimethylolomelamine; acetogenins (especially bulatacin and bulatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL); beta-
  • dynemycin including dynemycin A; a esperamycin; as well as neocarzinostatin chromophore and chromophores of related chromoprotein antibiotics), aclacinomisins, actinomycin, autramycin, azaserin, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorrubicin, 6-diazo-5-oxo- L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino- doxorubicin and deoxy doxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mit
  • Chimeric antibody refers to antibodies which comprise heavy and light chain variable region sequences from one species and constant region sequences from another species, such as antibodies having murine heavy and light chain variable regions linked to human constant regions.
  • Compete means that a first antibody binds to an epitope of a protein (e.g., CD22) in a manner sufficiently similar to the binding of a second antibody, such that the result of binding of the first antibody with its epitope is detectably decreased in the presence of the second antibody compared to the binding of the first antibody in the absence of the second antibody.
  • a first antibody can inhibit the binding of a second antibody to its epitope without that second antibody inhibiting the binding of the first antibody to its respective epitope.
  • each antibody detectably inhibits the binding of the other antibody with its epitope or ligand, whether to the same, greater, or lesser extent, the antibodies are said to “Cross-compete” with each other for binding of their respective epitope(s).
  • antibodies that compete or cross-compete bind to the same or overlapping epitopes. Regardless of the mechanism by which such competition or cross-competition occurs (e.g., steric hindrance, conformational change, or binding to a common epitope, or portion thereof), the skilled artisan would appreciate that such competing and/or cross-competing antibodies are encompassed and can be useful for the methods and/or compositions provided herein.
  • Conjugated means two entities are associated, preferably with sufficient affinity that a therapeutic/diagnostic benefit of the association between the two entities is realized.
  • the association between the two entities can be either direct or via a linker, such as a polymer linker.
  • Conjugated can include covalent or noncovalent bonding as well as other forms of association, such as entrapment, e.g., of one entity on or within the other, or of either or both entities on or within a third entity, such as a micelle.
  • Complementary refers to the capacity for precise pairing between two nucleotides or two sets of nucleotides.
  • complementary is a term that characterizes an extent of hydrogen bond pairing that brings about binding between two nucleotides or two sets of nucleotides. For example, if a base at one position of an oligonucleotide is capable of hydrogen bonding with a base at the corresponding position of a target nucleic acid (e.g., an mRNA), then the bases are considered to be complementary to each other at that position.
  • a target nucleic acid e.g., an mRNA
  • Base pairings may include both canonical Watson-Crick base pairing and non- Watson-Crick base pairing (e.g., Wobble base pairing and Hoogsteen base pairing).
  • adenosine-type bases are complementary to thymidine- type bases (T) or uracil-type bases (U)
  • cytosine-type bases are complementary to guanosine-type bases (G)
  • universal bases such as 3-nitropyrrole or 5-nitroindole can hybridize to and are considered complementary to any A, C, U, or T.
  • Inosine (I) has also been considered in the art to be a universal base and is considered complementary to any A, C, U or T.
  • a “conservative amino acid substitution” refers to an amino acid substitution that does not alter the relative charge or size characteristics of the protein in which the amino acid substitution is made.
  • Variants can be prepared according to methods for altering polypeptide sequence known to one of ordinary skill in the art such as are found in references which compile such methods, e.g. Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Fourth Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2012, or Current Protocols in Molecular Biology, F.M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York.
  • amino acids include substitutions made amongst amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.
  • Cross-reactive As used herein and in the context of a targeting agent (e.g., antibody), the term “cross-reactive,” refers to a property of the agent being capable of specifically binding to more than one antigen of a similar type or class (e.g., antigens of multiple homologs, paralogs, or orthologs) with similar affinity or avidity.
  • a targeting agent e.g., antibody
  • cross-reactive refers to a property of the agent being capable of specifically binding to more than one antigen of a similar type or class (e.g., antigens of multiple homologs, paralogs, or orthologs) with similar affinity or avidity.
  • Cytotoxic agent refers to a substance that inhibits or prevents a cellular function and / or causes cell death or destruction.
  • agents include, e.g., radioactive isotopes (e.g., At211, 1131, 1125, Y90, Rel86, Rel88, Sml53, Bi212, P32, Pb212 and radioactive isotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin, or other intercalating agents); growth inhibitory agents; enzymes and fragments thereof, such as nucleolytic enzymes; antibiotics; toxins such as small molecule toxins or enzymatically active toxins of
  • an effective amount refers to the amount of each active agent (e.g., anti-PRAME/HLA-A2 antibody) required to confer a desired effect (e.g., a therapeutic effect on the subject), either alone or in combination with one or more other active agents.
  • the therapeutic effect is reduced PRAME/HLA- A2 level or activity and/or alleviated disease conditions (e.g., treatment of a PRAME- expressing cancer or reduction and/or PRAME-expressing tumor size).
  • Framework refers to the remaining sequences of a variable region minus the CDRs. Because the exact definition of a CDR sequence can be determined by different systems, the meaning of a framework sequence is subject to correspondingly different interpretations.
  • the six CDRs also divide the framework regions on the light chain and the heavy chain into four sub-regions (FR1, FR2, FR3 and FR4) on each chain, in which CDR1 is positioned between FR1 and FR2, CDR2 between FR2 and FR3, and CDR3 between FR3 and FR4.
  • a framework region represents the combined FRs within the variable region of a single, naturally occurring immunoglobulin chain.
  • a FR represents one of the four sub-regions, and FRs represents two or more of the four sub-regions constituting a framework region.
  • Human heavy chain and light chain acceptor sequences are known in the art. In one embodiment, the acceptor sequences known in the art may be used in the antibodies disclosed herein.
  • Human antibody The term "human antibody”, as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences.
  • the human antibodies of the disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3.
  • the term "human antibody”, as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
  • Humanized antibody refers to antibodies which comprise heavy and light chain variable region sequences from a non-human species (e.g., a mouse) but in which at least a portion of the VH and/or VL sequence has been altered to be more "human-like", i.e., more similar to human germline variable sequences.
  • a non-human species e.g., a mouse
  • humanized antibody is a CDR-grafted antibody, in which human CDR sequences are introduced into non-human VH and VL sequences to replace the corresponding nonhuman CDR sequences.
  • humanized anti-PRAME/HLA-A2 antibodies and antigen binding portions are provided.
  • Such antibodies may be generated by obtaining murine anti-PRAME/HLA-A2 monoclonal antibodies using traditional hybridoma technology followed by humanization using in vitro genetic engineering, such as those disclosed in Kasaian et al PCT publication No. WO 2005/123126 A2.
  • Humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity.
  • CDR complementary determining region
  • donor antibody such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity.
  • Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non- human residues.
  • the humanized antibody may comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences but are included to further refine and optimize antibody performance.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody optimally will comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region or domain
  • Antibodies may have Fc regions modified as described in WO 99/58572.
  • Other forms of humanized antibodies have one or more CDRs (one, two, three, four, five, six) which are altered with respect to the original antibody, which are also termed one or more CDRs derived from one or more CDRs from the original antibody. Humanized antibodies may also involve affinity maturation.
  • humanization is achieved by grafting the CDRs (e.g., as shown in Table 1) into the human variable domains (e.g., IGKV1-NL1*O1 and IGHVl-3*01 human variable domain).
  • the anti-PRAME/HLA-A2 antibody of the present disclosure is a humanized variant comprising one or more amino acid substitutions (e.g., in the VH framework region) as compared with any one of the VHs listed in Table 1, and/or one or more amino acid substitutions (e.g., in the VL framework region) as compared with any one of the VLs listed in Table 1.
  • Isolated antibody An "isolated antibody”, as used herein, is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds PRAME/HLA-A2 is substantially free of antibodies that specifically bind antigens other than PRAME/HLA-A2).
  • An isolated antibody that specifically binds PRAME/HLA-A2 may, however, have crossreactivity to other antigens.
  • an isolated antibody may be substantially free of other cellular material and/or chemicals.
  • Kabat numbering The terms "Kabat numbering", “Kabat definitions” and “Kabat labeling” are used interchangeably herein. These terms, which are recognized in the art, refer to a system of numbering amino acid residues which are more variable (i.e. hypervariable) than other amino acid residues in the heavy and light chain variable regions of an antibody, or an antigen binding portion thereof (Kabat et al. (1971) Ann. NY Acad, Sci. 190:382-391 and, Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242).
  • the hypervariable region ranges from amino acid positions 31 to 35 for CDR1, amino acid positions 50 to 65 for CDR2, and amino acid positions 95 to 102 for CDR3.
  • the hypervariable region ranges from amino acid positions 24 to 34 for CDR1, amino acid positions 50 to 56 for CDR2, and amino acid positions 89 to 97 for CDR3.
  • Multispecific Antibody refers to a polypeptide or a complex (e.g., two or more covalently linked polypeptides) that includes at least two different immunoglobulin variable domains or at least two different sites, e.g., paratopes, that specifically bind to one or more antigens.
  • a multispecific antibody is a polypeptide that includes at least two different sites in which each site binds to a different epitope of the same antigen.
  • a multispecific antibody is a polypeptide that includes at least two different sites in which each site binds to a different antigen.
  • an antigen to which a site of a multispecific antibody specifically binds is a complex (e.g., a peptide complexed with a single chain HLA- A2 molecule).
  • PRAME PRAME, or PReferentially expressed Antigen in MElanoma, is a cancer- associated protein. Under normal conditions, PRAME is expressed intracellularly only in germline cells (e.g., sperm cells). It has been reported to function as a repressor of retinoic acid signaling. PRAME expression has been observed in a number of cancers, including but not limited to melanoma, leukemia, breast cancer, and lung cancer. In cancer cells, PRAME is processed by the proteosome such that fragments of the protein are presented on MHC-I molecules on the cancer cell surface, forming PRAME/HLA-A2 complexes in humans.
  • Recombinant antibody is intended to include all antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell (described in more details in this disclosure), including, for example, antibodies isolated from a recombinant, combinatorial human antibody library (Hoogenboom H. R., (1997) TIB Tech. 15:62-70; Azzazy H., and Highsmith W. E., (2002) Clin. Biochem. 35:425-445; Gavilondo J. V., and Larrick J. W.
  • recombinant human antibodies are provided herein.
  • such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences.
  • such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.
  • One embodiment of the disclosure provides fully human antibodies capable of binding human PRAME/HLA-A2 which can be generated using techniques well known in the art, such as, but not limited to, using human Ig phage libraries such as those disclosed in Jermutus et al., PCT publication No. WO 2005/007699 A2.
  • Selective refers to the ability of a molecule to produce an effect (e.g., inhibit, antagonize, agonize, etc.) in relation to its target molecule compared to a reference molecule.
  • a molecule that selectively inhibits its target molecule means that this molecule is capable of inhibiting its target molecule to a degree that is distinguishable from a reference molecule in an inhibition assay or other inhibitory context.
  • the term, “selectively inhibits”, refers to the ability of the inhibitor to inhibit its target molecule with a degree that is distinguishable from a reference molecule that is not substantially inhibited in an inhibition assay, e.g., to an extent that permit selective inhibition of the target molecule, as described herein.
  • the signal produced by inhibiting the target molecule can be measured.
  • the half maximal inhibitor concentration for the target molecule and the reference molecule can be calculated.
  • a molecule described herein selectively binds to a target molecule.
  • a molecule described herein selectively binds PRAME/HLA-A2.
  • a molecule described herein directs a T cell to target a PRAME/HLA-A2 expressing cell. In some embodiments, a molecule described herein binds to PRAME/HLA-A2 to induce killing of PRAME/HLA-A2 expressing cells, e.g., PRAME/HLA-A2 expressing tumor cells.
  • the term “specifically binds” refers to the ability of a molecule to bind to a binding partner with a degree of affinity or avidity that enables the molecule to be used to distinguish the binding partner from an appropriate control in a binding assay or other binding context.
  • the term, “specifically binds”, refers to the ability of the antibody to bind to a specific antigen with a degree of affinity or avidity, compared with an appropriate reference antigen or antigens, that enables the antibody to be used to distinguish the specific antigen from others, as described herein.
  • an antibody specifically binds to a target if the antibody has a KD for binding the target of at least about 10' 4 M, 10' 5 M, 10' 6 M, 10' 7 M, 10' 8 M, 10' 9 M, 10’ 10 M, 10' 11 M, 10' 12 M, 10’ 13 M, or less. In some embodiments, an antibody specifically binds a PRAME peptide bound by HLA-A2.
  • Subject refers to a mammal.
  • a subject is a human.
  • a subject is a patient, e.g., a human patient that has or is suspected of having a disease.
  • the subject is a human patient who has or is suspected of having a PRAME-expressing cancer or tumor and/or one or more conditions arising as a result of a PRAME-expressing cancer or tumor.
  • treating refers to the application or administration of a composition including one or more active agents (e.g., anti- PRAME/HLA-A2 antibodies) to a subject, who has a target disease or disorder (e.g., cancer expressing PRAME), a symptom of the disease/disorder, or a predisposition toward the disease/disorder, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disorder, the symptom of the disease, or the predisposition toward the disease or disorder.
  • Alleviating a target disease/disorder includes delaying or preventing the development or progression of the disease or reducing disease severity.
  • the anti-PRAME/HLA-A2 antibody is an antibody specific for PRAME/HLA-A2.
  • the anti- PRAME/HLA-A2 antibody described herein specifically binds to any extracellular epitope of a PRAME/HLA-A2 or an epitope that becomes exposed to an antibody.
  • PRAME PReferentially expressed Antigen in MElanoma
  • PRAME is one of the most highly expressed cancer-associated antigens. Under homeostatic conditions, PRAME is expressed intracellularly only in the testis or placenta and is thus categorized as a cancer-testis antigen. The normal function of PRAME is not fully understood, but it is thought to promote cancer cell proliferation, survival, and immune evasion. While PRAME is normally only expressed intracellularly, in cancer cells it is broken down into peptides and peptide fragments, which are then bound by HLA molecules for display on the cancer cell surface.
  • PRAME PReferentially expressed Antigen in MElanoma
  • PRAME expression is a protein found to be expressed by cells in a number of cancers, including melanoma, leukemia, breast cancer, lung cancer, and others. In healthy conditions, PRAME expression is limited to germline cells (e.g., sperm cells) and is not expressed in somatic cells.
  • PRAME is an intracellular protein thought to function as a repressor of retinoic acid signaling, but in cancer cells it is processed by the proteosome such that PRAME peptides are presented on MHC or HLA molecules on the cancer cell surface, forming a PRAME/MHC complex (e.g., a PRAME/MHC-I complex) or a PRAME/HLA complex (e.g., a PRAME/HLA-A2 complex).
  • PRAME/MHC complex e.g., a PRAME/MHC-I complex
  • PRAME/HLA complex e.g., a PRAME/HLA-A2 complex.
  • Such complexes can be recognized by immune cells, including T effector cells.
  • an anti-PRAME/HLA-A2 antibody described herein specifically binds to a human PRAME peptide bound by HLA-A2.
  • Exemplary amino acids sequence of human PRAME is set forth in NCBI Accession Numbers CAG30435.1, AAH39731.1, AFX65484.1, AFX65485.1, AFX65486.1, AFX65487.1, KAI2596882.1, KAI2596883.1, KAI2596884.1, KAI2596885.1, or KAI2596886.1.
  • An exemplary amino acid sequence of human PRAME is set forth in SEQ ID NO: 20.
  • an anti- PRAME/HLA-A2 antibody described herein specifically binds to a PRAME peptide comprising the amino acid sequence consists of SLLQHLIGL (SEQ ID NO: 19).
  • the present disclosure provides antibodies that specifically binds members of a set of complexes of peptides bound by HLA-A2, each complex comprising a different peptide bound by HLA-A2, wherein each peptide has an amino acid sequence defined by a formula set forth as X56X57X58X59HLIGX60 (SEQ ID NO: 18).
  • each peptide has an amino acid sequence defined by a formula set forth as X56X57X58X59HLIGX60 (SEQ ID NO: 18).
  • X56 is S
  • X58 is L
  • X59 Q
  • Xeo is L
  • X57 is M or L.
  • X56 when X56 is S, X57 is L, X59 is Q, and Xeo is L, X58 is D, M, W, Y, L, N, Q, F, H, A, or T.
  • X56 when X56 is S, X57 is L, X58 is L, and Xeo is L, X59 is E, P, N, Q, S, G, K, T, or A.
  • X56 when X56 is S, X57 is L, X58 is L, and X59 is Q, Xeo is Y, I, T, L, or A.
  • X53 when X53 is S, X54 is L, X56 is Q, X57 is H, and X58 is L, X55 is Y, M, L, F, H, D, or W. In some embodiments, when X53 is S, X54 is L, X55 is L, X57 is H, and X58 is L, X56 is D, E, P, N, G, S, Q, T, K, or A. In some embodiments, when X53 is S, X54 is L, X55 is L, X56 is Q, and X58 is L, X57 is W, S, A, N, H, G, T, or S. In some embodiments, when X53 is S, X54 is L, X55 is L, X56 is Q, and X57 is H, X58 is Y, T, L, or A.
  • At least one member of the set of complexes is a PRAME peptide bound by HLA-A2.
  • the PRAME peptide comprises the amino acid sequence consisting of SLLQHLIGL (SEQ ID NO: 19).
  • the present disclosure also provides an antibody obtained by a process comprising:(l) exposing members of a set of complexes, each complex comprises a different peptide bound by HLA-A2, wherein each peptide has an amino acid sequence defined by a formula set forth as X56X57X58X59HLIGX60 (SEQ ID NO: 18), to a library of antibodies under conditions in which at least one antibody of the library that specifically binds to the members of the set of complexes is detected; (2) obtaining the sequence of a heavy chain variable domain of an antibody detected in step (1) that specifically binds the members of the set of complexes; and (3) producing an antibody having at least a heavy chain complementarity determining region 3 (HC CDR3) of the heavy chain variable domain of step (2), thereby obtaining the antibody that specifically binds to the members of the set of complexes.
  • HC CDR3 heavy chain complementarity determining region 3
  • the process further comprising (4) confirming that the antibody produced by step (3) binds PRAME/HLA-A2 complex (e.g., a complex comprising SLLQHLIGL (SEQ ID NO: 19 bound by HLA-A2).
  • PRAME/HLA-A2 complex e.g., a complex comprising SLLQHLIGL (SEQ ID NO: 19 bound by HLA-A2).
  • a need for means for binding a PRAME-HLA-A2 complex exists for treating cancers expressing a PRAME peptide bound by HLA-A2 on the cell surface.
  • the present disclosure provides a means for specifically binding a peptide/HLA-A2 complex, wherein the peptide/HLA-A2 complex is a member of a set of complexes of peptides bound by HLA-A2, each complex comprising a different peptide bound by HLA-A2, wherein each peptide has an amino acid sequence defined by a formula set forth as X56X57X58X59HLIGX60 (SEQ ID NO: 18).
  • the present disclosure provides a means for specifically binding a PRAME/HLA-A2 complex, wherein the PRAME/HLA-A2 complex comprises a PRAME peptide comprising the amino acid sequence of SLLQHLIGL (SEQ ID NO: 19) bound by HLA-A2.
  • the means for specifically binding a peptide/HLA-A2 complex e.g., peptide X56X57X58X59HLIGX60 (SEQ ID NO: 18) bound by HLA-A2
  • the means for specifically binding a PRAME/HLA-A2 complex e.g., PRAME peptide SLLQHLIGL (SEQ ID NO: 19) bound by HLA-A2
  • a PRAME/HLA-A2 complex e.g., PRAME peptide SLLQHLIGL (SEQ ID NO: 19) bound by HLA-A2
  • an anti-PRAME/HLA-A2 antibody described herein e.g., an antibody described in Table 1 or equivalents thereof.
  • the present disclosure also provides an antibody obtained by a process comprising: (1) exposing members of a set of complexes, each complex comprises a different peptide bound by HLA-A2, wherein each peptide has an amino acid sequence defined by a formula set forth as X53X54X55X56X57LIGX58, to a library of antibodies under conditions in which at least one antibody of the library that specifically binds to the members of the set of complexes is detected; (2) obtaining the sequence of a heavy chain variable domain of an antibody detected in step (1) that specifically binds the members of the set of complexes; and (3) producing an antibody having at least a heavy chain complementarity determining region 3 (HC CDR3) of the heavy chain variable domain of step (2), thereby obtaining an antibody that specifically binds to the members of the set of complexes.
  • HC CDR3 heavy chain complementarity determining region 3
  • the process further comprising (4) confirming that the antibody produced by step (3) binds PRAME/HLA-A2 complex (e.g., a complex comprising SLLQHLIGL (SEQ ID NO: 19 bound by HLA-A2).
  • PRAME/HLA-A2 complex e.g., a complex comprising SLLQHLIGL (SEQ ID NO: 19 bound by HLA-A2).
  • a need for means for binding a PRAME-HLA-A2 complex exists for treating cancers expressing a PRAME peptide bound by HLA-A2 on the cell surface.
  • the present disclosure provides a means for specifically binding a peptide/HLA-A2 complex, wherein the peptide/HLA-A2 complex is a member of a set of complexes of peptides bound by HLA-A2, each complex comprising a different peptide bound by HLA-A2, wherein each peptide has an amino acid sequence defined by a formula set forth as X53X54X55X56X57LIGX58.
  • the present disclosure provides a means for specifically binding a PRAME/HLA-A2 complex, wherein the PRAME/HLA-A2 complex comprises a PRAME peptide comprising the amino acid sequence of SLLQHLIGL (SEQ ID NO: 19) bound by HLA-A2.
  • the means for specifically binding a peptide/HLA-A2 complex e.g., peptide X53X54X55X56X57LIGX58 bound by HLA-A2
  • the means for specifically binding a PRAME/HLA-A2 complex e.g., PRAME peptide SLLQHLIGL (SEQ ID NO: 19) bound by HLA-A2
  • a PRAME/HLA-A2 complex e.g., PRAME peptide SLLQHLIGL (SEQ ID NO: 19) bound by HLA-A2
  • an anti-PRAME/HLA-A2 antibody described herein e.g., an antibody described in Table 1 or equivalents thereof.
  • the anti-PRAME/HLA-A2 antibody described herein specifically binds to an epitope on human PRAME bound by HLA-A2.
  • the anti-PRAME/HLA-A2 antibody described herein may bind to a fragment of a human PRAME/HLA-A2.
  • the fragment of PRAME/HLA-A2 may be between about 5 and about 425 amino acids, between about 10 and about 400 amino acids, between about 50 and about 350 amino acids, between about 100 and about 300 amino acids, between about 150 and about 250 amino acids, between about 200 and about 300 amino acids, or between about 75 and about 150 amino acids in length.
  • the fragment may comprise a contiguous number of amino acids from PRAME/HLA-A2.
  • the anti-PRAME/HLA-A2 antibodies described herein are affinity matured clones.
  • an anti-PRAME/HLA-A2 antibody specifically binds a PRAME/HLA-A2 (e.g., a human PRAME/HLA-A2) with binding affinity (e.g., as indicated by KD) of at least about 10' 4 M, 10' 5 M, 10' 6 M, 10' 7 M, 10' 8 M, 10’ 9 M, 10' 10 M, 10' 11 M, 10' 12 M, IO’ 13 M, or less.
  • the anti-PRAME/HLA-A2 antibodies of the present disclosure can bind to a PRAME/HLA-A2 protein complex (e.g., human PRAME/HLA-A2) with an affinity between 5 pM and 500 nM, e.g., between 50 pM and 100 nM, e.g., between 500 pM and 50 nM.
  • a PRAME/HLA-A2 protein complex e.g., human PRAME/HLA-A2 protein complex
  • the disclosure also includes antibodies that compete with any of the antibodies described herein for binding to a PRAME/HLA-A2 protein complex (e.g., human PRAME/HLA-A2) and that have an affinity of 100 nM or lower (e.g., 80 nM or lower, 50 nM or lower, 20 nM or lower, 10 nM or lower, 500 pM or lower, 50 pM or lower, or 5 pM or lower).
  • the affinity and binding kinetics of the anti- PRAME/HLA-A2 antibody can be tested using any suitable method including but not limited to biosensor technology (e.g., OCTET or BIACORE).
  • the anti- PRAME/HLA-A2 antibodies described herein bind to PRAME/HLA-A2 with a KD of submicromolar range.
  • Binding affinity can be determined by a variety of methods including equilibrium dialysis, equilibrium binding, gel filtration, ELISA, surface plasmon resonance (SPR), florescent activated cell sorting (FACS) or spectroscopy (e.g., using a fluorescence assay).
  • Exemplary conditions for evaluating binding affinity are in HBS-P buffer (10 mM HEPES pH7.4, 150 mM NaCl, 0.005% (v/v) surfactant P20) and PBS buffer (lOmM PO4-3, 137mM NaCl, and 2.7mM KC1). These techniques can be used to measure the concentration of bound proteins as a function of target protein concentration.
  • the concentration of bound protein [[Bound]]
  • the concentration of bound protein is generally related to the concentration of free target protein ([[Free]]) by the following equation:
  • an antibody is a full-length IgG, a Fab fragment, a F(ab') fragment, a F(ab’)2 fragment, a scFv, or a Fv.
  • the antibody comprises a heavy chain constant region of the isotype IgGl, IgG2, IgG3, or IgG4.
  • an anti-PRAME- HEA-A2 antibody of the present disclosure comprises a HC CDR1 having the amino acid sequence of X1X2X3X4X5X6X7, wherein Xi is M, S, or absent; X2 is D, G, N, S, or absent; X3 is G, S, N, or T; X4 is D, F, N, H, or Y; X5 is F, S, W, or Y; Xe is W or absent; and X7 is N or S.
  • an anti-PRAME-HEA-A2 antibody of the present disclosure comprises a HC CDR2 having the amino acid sequence of X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16, wherein Xi is E, F, Y, or W; X 2 is I, M, or V; X 3 is F, Y, or N; X 4 is D, H, N, Y, or P; X 5 is S, T, or N; X 6 is G, E, or S; X 7 is N, S, T, or G; X 8 is T or Y; X 9 is N, S, Y, or T; X10 is Y or G; Xu is N or Y; X12 is P or A; X13 is S or Q; X i4 is E or K; X15 is K or F; and Xi6 is S, T, or Q.
  • an anti-PRAME- HEA-A2 antibody of the present disclosure comprises a HC CDR3 having the amino acid sequence 01X1X2X3X4X5X6X7X8X9X10X11X12, wherein Xi is E or absent; X2 is E, G, or absent; X 3 is D, Q, T, or W; X 4 is D, E, I, M, T, or Y ; X 5 is I, L, M, or N ; X 6 is I, L, R, or W, X 7 is G, N, or R ; X 8 is A, G, F ; X 9 is A, F, G, H, or V; X10 is F, I, or E; Xu is D or G; X12 is D, G, H, I, P, or Y.
  • an anti-PRAME- HLA-A2 antibody of the present disclosure comprises a LC CDR1 having the amino acid sequence of RASX4X5ISX8WLA (SEQ ID NO: 186), wherein X4 is Q or P; X5 is G or D; and X 8 is N, R, or S.
  • an anti-PRAME- HLA-A2 antibody of the present disclosure comprises a LC CDR2 having the amino acid sequence of X1X2SX4LX6X7, wherein Xi is A, T, or V; X2 is A or V; X4 is N or S; Xi> is H or Q; and H7 is G or S.
  • an anti-PRAME- HLA-A2 antibody of the present disclosure comprises a LC CDR3 having the amino acid sequence of QQX3NX5FPX8T (SEQ ID NO: 187), wherein X 3 is A or T; X 5 is N, R, or S; X 8 is F, L, or W.
  • an anti- PRAME/HLA-A2 antibody comprises a HC CDR1 having the amino acid sequence of X1X2X3X4X5WX6, wherein Xi is S or N; X2 is Y, N, or G; X3 is F, N or S; X4 is H or absent; X5 is W, Y or absent; and Xi> is N or S.
  • Xi of HC CDR1 when Xi of HC CDR1 is N, X2 is N and X5 is W.
  • X4 of CDR1 when X4 of CDR1 is H, Xi is S and X5 is Y.
  • Xi of HC CDR1 when Xi of HC CDR1 is S, X2 is Y or G, and X3 is F or S. In some embodiments, when X3 of HC CDR1 is F, Xi is S and X5 is absent.
  • an anti- PRAME/HLA-A2 antibody comprises a HC CDR3 having the amino acid sequence of X11X12X13X14IRGX15X16X17X18X19, wherein Xu is D or E; X12 is G, W, or absent; X13 is T,
  • X14 is M or L
  • X15 is V, A or H
  • Xi6 is G or absent
  • X17 is L or F
  • Xis is G or D
  • X19 is Y, P, I, or D.
  • Xu of HC CDR3 is E
  • X12 is G or W
  • X15 is A or H
  • X19 is P, I, or D.
  • X12 of HC CDR3 is W
  • X13 is D or
  • X15 is A or H
  • X19 is I or D
  • X14 of HC CDR3 is M
  • Xu is D or E
  • X12 is G or absent
  • X15 is V or A
  • X19 is Y or P.
  • X14 is L
  • X13 is D or E
  • X15 is A or H
  • X19 is I or D.
  • an anti- PRAME/HLA-A2 antibody comprises a LC CDR1 having the amino acid sequence of RASX20X21ISX22WLA (SEQ ID NO: 4), wherein X20 is Q or P, X21 is G or D, and X22 is S, R, or N.
  • an anti- PRAME/HLA-A2 antibody comprises a LC CDR2 having the amino acid sequence of X23ASSLQX24 (SEQ ID NO: 5), wherein X23 is A, T or V, and X24 is S or G. In some embodiments, when X23 of LC CDR2 is A, X24 is S or G.
  • an anti- PRAME/HLA-A2 antibody comprises a LC CDR3 having the amino acid sequence of QQX25NX26FPX27T (SEQ ID NO: 6), wherein X 25 is T or A; X26 is N or S; and X27 is W or L.
  • SEQ ID NO: 6 QQX25NX26FPX27T
  • Xi of HC CDR1 of an anti-PRAME/HLA-A2 antibody is S
  • X2 is Y or G
  • X3 is F or S
  • X5 is T or absent
  • Xi> is S or N
  • X7 is E, Y or F
  • Xs is
  • X9 is S or T
  • X10 is S, N, or T
  • Xu is D or E
  • X12 is W or absent
  • X13 is T, D, or E
  • X14 is M or L
  • X15 is V, A or H
  • Xi6 is absent
  • X17 is L or F
  • Xis is G or D
  • X19 is Y, I, or D.
  • X23 is A or V
  • X24 is S or G
  • X25 is T or A
  • X27 is L.
  • X7 of HC CDR2 of an anti-PRAME/HLA-A2 antibody is F
  • Xi is S or N
  • X2 is G
  • X3 is S, F, or N
  • X4 is H or absent
  • X5 is Y
  • Xe is S or N
  • X 8 is Y
  • X9 is T or S
  • X10 is T, N, or S
  • Xu E or D
  • X12 is W
  • X13 is E, D, or T
  • X14 is L or M
  • X15 is H, A, or V
  • Xi6 is absent or G
  • X17 is F
  • Xis is D or G
  • X19 is D.
  • Xi is N or S
  • X2 is N, Y, or G
  • X3 is Y, F, or S
  • X4 is H or absent
  • X5 is W, Y, or absent
  • Xi> is S
  • X7 is Y or F
  • X 8 is Y or D
  • X9 is S or T
  • X10 is T or N
  • X12 is G or W
  • X13 is T, D, or E
  • X14 is M or L
  • X15 is A or H
  • Xi6 is G or absent
  • X17 is F
  • Xis is D
  • X19 is P
  • an antibody comprises a HC CDR1, a HC CDR2, a HC CDR3, a LC CDR1, a LC CDR2, and a LC CDR3 according to paragraphs [00101]-[00114].
  • the antibody of paragraph [00115] further comprises a framework region 1 (FR1) comprising the amino acid sequence QVQLX40X41SGX42X43X44X45KPX46X47X48X49X50X51X52CX53X54SGX55X56X57X58 (SEQ ID NO: 188), wherein X40 is Q or V; X41 is E or Q; X42 is A or P; X43 is E or G; X44 is L or V;
  • FR1 framework region 1
  • the antibody of paragraph [00115] further comprises a framework region 2 (FR2) comprising the amino acid sequence WX59RX60X61PGX62GLX63WIG (SEQ ID NO: 189), wherein X59 is I or V; X 60 is Q or R; X 6i is P or T; X62 is K or R; X63 is E or G.
  • FR2 framework region 2
  • the antibody of paragraph [00115] further comprises a framework region 3 (FR3) comprising the amino acid sequence RVTX64X6 5 X66X67X 6 8X69X70X71X72X73X74X7 5 X76X77X78SX79X80X81X82DTAX83YYCAR (SEQ ID NO: 190), wherein X64 is I, L, or M; Xes is S or T; Xee is I, L, R, or V; X67 is D or N; Xes is T, K, or P; X69 is P or S; X70 is I or K; X71 isN or S; X72 is H, Q, or T; X73 is A or F; X74 is S or Y; X75 is L or M; X76 is E, K, M, N, or R; X77 is L or M; X78 is N, S, T, or R; X79 is L or V
  • FR3 framework region 3
  • the antibody of paragraph [00115] further comprises a framework region 3 (FR3) comprising the amino acid sequence WGX84GTX85VTVSS (SEQ ID NO: 191), wherein Xs4 is R or Q and Xss is L or M.
  • FR3 framework region 3
  • an anti- PRAME/HLA-A2 antibody of the present disclosure comprises a HC CDR1 having the amino acid sequence of X1X2X3X4X5WX6, a HC CDR2 having the amino acid sequence of SEQ ID NO: 2, a HC CDR3 having the amino acid sequence of X11X12X13X14IRGX15X16X17X18X19, a LC CDR1 having the amino acid sequence of SEQ ID NO: 4, a LC CDR2 having the amino acid sequence of SEQ ID NO: 5, and a LC CDR3 having the amino acid sequence of SEQ ID NO: 6.
  • an anti-PRAME/HLA-A2 antibody comprises a variable heavy domain (VH) with a framework region 1 (FR1) having the amino acid sequence of SEQ ID NO: 7, a framework region 2 (FR2) having the amino acid sequence of SEQ ID NO: 8, a framework region 3 (FR3) having the amino acid sequence of SEQ ID NO: 9, and a framework region 4 (FR4) having the amino acid sequence of SEQ ID NO: 10.
  • VH variable heavy domain
  • an anti-PRAME/HLA-A2 antibody comprises a VH domain with a FR1 having the amino acid sequence of QVQLQESGPGLVKPSX28TLSLTCX29VSGX30SX31X32 (SEQ ID NO: 7), wherein X 28 is G or E; X29 is T or A; X30 is G or D; X31 is I or V; and X32 is N or S.
  • an anti-PRAME/HLA-A2 antibody comprises a VH domain with a FR2 having the amino acid sequence of WX33RQPPGKGLEWIG (SEQ ID NO: 8), wherein X33 is I or V.
  • an anti-PRAME/HLA-A2 antibody comprises a VH domain with a FR4 having the amino acid sequence of WGQGTX39VTVSS (SEQ ID NO: 10), wherein X39 is L or M.
  • an antibody further comprises a light chain complementarity determining region 1 (LC CDR1) comprising the amino acid sequence RASQGISSWLA (SEQ ID NO: 24); a light chain complementarity determining region 2 (LC CDR2) comprising the amino acid sequence X52ASSLQS (SEQ ID NO: 14), wherein X52 is A or V; and a light chain complementarity determining region 3 (LC CDR3) comprising the amino acid sequence QQX53NX54FPLT (SEQ ID NO: 15), wherein X53 is T or A, and X54 is N or S.
  • LC CDR1 comprising the amino acid sequence RASQGISSWLA
  • LC CDR2 comprising the amino acid sequence X52ASSLQS (SEQ ID NO: 14), wherein X52 is A or V
  • LC CDR3 comprising the amino acid sequence QQX53NX54FPLT (SEQ ID NO: 15), wherein X53 is T or A, and X54 is N or S.
  • X44 is S
  • X40 is S
  • X41 is Y
  • X42 is F or Y
  • X43 is S
  • X45 is N or Y
  • X46 is D or E
  • X47 is W
  • X49 is L
  • X50 is H
  • X51 is L
  • I is or F
  • X53 is V
  • X54 is T or A
  • X55 is N or S.
  • X51 is F
  • X40 is S
  • X41 is Y
  • X42 is F or Y
  • X43 is S
  • X44 is T or S
  • X45 is Y or N
  • X46 is E
  • X47 is W
  • X48 is E
  • X50 is H
  • X52 is D
  • X53 is V
  • X54 is A
  • X55 is S.
  • X40 when X40 is T, X41 is F, X42 is F, X43 is N, X44 is T, X45 is N, X46 is D, X47 is T, X48 is M, X49 is absent, X50 is V, X51 is I, X52 is G, X53 is A, X54 is T, and X55 is N.
  • X40 when X40 is S, X41 is Y, X42 is F, X43 is N, X44 is T, X45 is N, X46 is D, X47 is T, X48 is M, X49 is absent, X50 is V, X51 is L, X52 is G, X53 is A, X54 is T, and X55 is N.
  • the disclosure provides an antibody comprising a heavy chain complementarity determining region 1 (HC CDR1) comprising the amino acid sequence X40X41X42WWS, wherein X40 is S or N, X41 is S, G or N, and X42 is N or D; a heavy chain complementarity determining region 2 (HC CDR2) comprising the amino acid sequence EX43YX44SGSTX45YNPSLKX46 (SEQ ID NO: 200), wherein X43 is I or V; X44 is H or N; X 45 is N or S; and X46 is S or T; and a heavy chain complementarity determining region 3 (HC CDR3) comprising the amino acid sequence EGTMIRGAGFDP (SEQ ID NO: 201).
  • an antibody further comprises a light chain complementarity determining region 1 (LC CDR1) comprising the amino acid sequence RASX47X48ISX49WLA (SEQ ID NO: 202), wherein X47 is Q or
  • LC CDR2 light chain complementarity determining region 2
  • LC CDR3 light chain complementarity determining region 3
  • X41 is S
  • X40 is S
  • X42 is N
  • X43 is I or V
  • X44 is H or N
  • X45 is N or S
  • X46 is S or T
  • X47 is Q or P
  • X48 is G or D
  • X49 is R or N
  • X50 is A
  • X51 is H or Q
  • X52 is S.
  • X45 is S
  • X40 is S
  • X41 is G or N
  • X42 is D or N
  • X43 is I
  • X44 is H
  • X46 is S
  • X47 is Q
  • X48 is G
  • X49 is S
  • X50 is A
  • X51 is W
  • X52 is S or R.
  • X40 is S
  • X41 is N
  • X42 is N
  • X43 is I
  • X44 is H
  • X45 is S
  • X46 is S
  • X47 is Q
  • X48 is G
  • X49 is S
  • X50 is A
  • X51 is
  • X40 is N
  • X41 is N
  • X42 is N
  • X43 is I
  • X44 is H
  • X45 is N
  • X46 is S
  • X47 is Q
  • X48 is G
  • X49 is S
  • X50 is T
  • X51 is Q
  • X52 is S.
  • an antibody is a full-length IgG, a Fab fragment, a F(ab') fragment, a F(ab’)2 fragment, a scFv, or a Fv.
  • an antibody comprises a heavy chain constant region of the isotype IgGl, IgG2, IgG3, or IgG4.
  • VH heavy chain variable domain
  • VL light chain variable domain
  • CDR sequences CDR sequences
  • heavy chain and light chain constant region sequences of non-limiting examples of anti-PRAME/HLA-A2 antibodies are provided in Table 1.
  • the present disclosure provides an antibody that specifically binds PRAME/HLA-A2 complex, wherein the improvement comprises one or more of the HC CDRs (e.g., HC CDR1, HC CDR2, or HC CDR3) amino acid sequences from any one of the anti-PRAME/HLA-A2 antibodies selected from Table 1.
  • the present disclosure provides an antibody that specifically binds PRAME/HLA-A2 complex, wherein the improvement comprises the HC CDR1, HC CDR2, and HC CDR3 as provided for any one of the antibodies elected from Table 1.
  • the present disclosure provides an antibody that specifically binds PRAME/HLA-A2 complex, wherein the improvement comprises one or more of the LC CDRs (e.g., LC CDR1, LC CDR2, or LC CDR3) amino acid sequences from any one of the anti-PRAME/HLA-A2 antibodies selected from Table 1.
  • the present disclosure provides an antibody that specifically binds PRAME/HLA-A2 complex, wherein the improvement comprises the LC CDR1, LC CDR2, and LC CDR3 as provided for any one of the anti-PRAME/HLA-A2 antibodies selected from Table 1.
  • the present disclosure provides an antibody that specifically binds PRAME/HLA-A2 complex, wherein the improvement comprises HC CDR1, HC CDR2, and HC CDR3, LC CDR1, LC CDR2, and LC CDR3 as provided for any one of the anti-PRAME/HLA-A2 antibodies selected from Table 1.
  • an anti-PRAME/HLA-A2 antibody of the present disclosure comprises one or more of the HC CDRs (e.g., HC CDR1, HC CDR2, or HC CDR3) amino acid sequences from any one of the anti-PRAME/HLA-A2 antibodies selected from Table 1.
  • an anti-PRAME/HLA-A2 antibody of the present disclosure comprises the HC CDR1, HC CDR2, and HC CDR3 as provided for any one of the antibodies elected from Table 1.
  • an anti-PRAME/HLA-A2 antibody of the present disclosure comprises one or more of the LC CDRs (e.g., LC CDR1, LC CDR2, or LC CDR3) amino acid sequences from any one of the anti-PRAME/HLA-A2 antibodies selected from Table 1.
  • an anti-PRAME/HLA-A2 antibody of the present disclosure comprise the LC CDR1, LC CDR2, and LC CDR3 as provided for any one of the anti-PRAME/HLA-A2 antibodies selected from Table 1.
  • the present disclosure provides an antibody that specifically binds PRAME/HLA-A2 complex, wherein the improvement comprises the HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 as provided for any one of the anti-PRAME/HLA-A2 antibodies selected from Table 1.
  • the anti-PRAME/HLA-A2 antibodies of the present disclosure comprises the HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 as provided for any one of the anti-PRAME/HLA-A2 antibodies selected from Table 1.
  • antibody heavy and light chain CDR3 domains may play a particularly important role in the binding specificity /affinity of an antibody for an antigen.
  • the anti-PRAME/HLA-A2 antibodies of the disclosure may include at least the heavy and/or light chain CDR3 as of any one of the anti-PRAME/HLA-A2 antibodies selected from Table 1.
  • the isolated anti-PRAME/HLA-A2 antibody comprises a heavy chain variable region that comprises a heavy chain CDR1 (HC CDR1), a heavy chain CDR2 (HC CDR2), and a heavy chain CDR3 (HC CDR3).
  • HC CDR1 heavy chain CDR1
  • HC CDR2 heavy chain CDR2
  • HC CDR3 heavy chain CDR3
  • a functional variant may contain one or more amino acid residue variations in the VH and/or VL, or in one or more of the HC CDRs and/or one or more of the LC CDRs as relative to the reference antibody, while retaining substantially similar binding and biological activities e.g., substantially similar binding affinity, binding specificity, inhibitory activity, anti-inflammatory activity, or a combination thereof) as the reference antibody.
  • any of the anti-PRAME/HLA-A2 antibodies of the disclosure have one or more CDRs (e.g., HC CDR or LC CDR) sequences substantially similar to any of the HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and/or LC CDR3 sequences from one of the anti-PRAME/HLA-A2 antibodies selected from Table 1.
  • CDRs e.g., HC CDR or LC CDR sequences substantially similar to any of the HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and/or LC CDR3 sequences from one of the anti-PRAME/HLA-A2 antibodies selected from Table 1.
  • the position of one or more CDRs along the VH (e.g., HC CDR1, HC CDR2, or HC CDR3) and/or VL (e.g., LC CDR1, LC CDR2, or LC CDR3) region of an antibody described herein can vary by one, two, three, four, five, or six amino acid positions so long as immuno specific binding to PRAME/HLA-A2 (e.g., human PRAME/HLA-A2) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the binding of the original antibody from which it is derived).
  • PRAME/HLA-A2 e.g., human PRAME/HLA-A2
  • the position defining a CDR of any antibody described herein can vary by shifting the N-terminal and/or C-terminal boundary of the CDR by one, two, three, four, five, or six amino acids, relative to the CDR position of any one of the antibodies described herein, so long as immuno specific binding to PRAME/HLA- A2 (e.g., human PRAME/HLA-A2) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the binding of the original antibody from which it is derived).
  • PRAME/HLA- A2 e.g., human PRAME/HLA-A2
  • substantially maintained for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the binding of the original antibody from which it is derived.
  • the length of one or more CDRs along the VH (e.g., HC CDR1, HC CDR2, or HC CDR3) and/or VL (e.g., LC CDR1, LC CDR2, or LC CDR3) region of an antibody described herein can vary (e.g., be shorter or longer) by one, two, three, four, five, or more amino acids, so long as immuno specific binding to PRAME/HLA-A2 (e.g., human PRAME/HLA-A2) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the binding of the original antibody from which it is derived).
  • PRAME/HLA-A2 e.g., human PRAME/HLA-A2
  • a HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and/or LC CDR3 described herein may be one, two, three, four, five or more amino acids shorter than one or more of the CDRs described herein (e.g., CDRS from any of the anti-PRAME/HLA-A2 antibodies selected from Table 1) so long as immuno specific binding to PRAME/HLA-A2 (e.g., human PRAME/HLA-A2) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% relative to the binding of the original antibody from which it is derived).
  • PRAME/HLA-A2 e.g., human PRAME/HLA-A2
  • a HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and/or LC CDR3 described herein may be one, two, three, four, five or more amino acids longer than one or more of the CDRs described herein (e.g., CDRS from any of the anti-PRAME/HLA-A2 antibodies selected from Table 1) so long as immuno specific binding to PRAME/HLA-A2 (e.g., human PRAME/HLA-A2) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% relative to the binding of the original antibody from which it is derived).
  • CDRS from any of the anti-PRAME/HLA-A2 antibodies selected from Table 1
  • PRAME/HLA-A2 e.g., human PRAME/HLA-A2
  • substantially maintained for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at
  • the amino portion of a HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and/or LC CDR3 described herein can be extended by one, two, three, four, five or more amino acids compared to one or more of the CDRs described herein (e.g., CDRS from any of the anti-PRAME/HLA-A2 antibodies selected from Table 1) so long as immuno specific binding to PRAME/HLA-A2 (e.g., human PRAME/HLA-A2) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% relative to the binding of the original antibody from which it is derived).
  • PRAME/HLA-A2 e.g., human PRAME/HLA-A2
  • the carboxy portion of a HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and/or LC CDR3 described herein can be extended by one, two, three, four, five or more amino acids compared to one or more of the CDRs described herein (e.g., CDRS from any of the anti-PRAME/HLA-A2 antibodies selected from Table 1) so long as immuno specific binding to PRAME/HLA-A2 (e.g., human PRAME/HLA-A2) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% relative to the binding of the original antibody from which it is derived).
  • PRAME/HLA-A2 e.g., human PRAME/HLA-A2
  • the amino portion of a HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and/or LC CDR3 described herein can be shortened by one, two, three, four, five or more amino acids compared to one or more of the CDRs described herein (e.g., CDRS from any of the anti-PRAME/HLA-A2 antibodies selected from Table 1) so long as immuno specific binding to PRAME/HLA-A2 (e.g., human PRAME/HLA-A2) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% relative to the binding of the original antibody from which it is derived).
  • the carboxy portion of a HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and/or LC CDR3 described herein can be shortened by one, two, three, four, five or more amino acids compared to one or more of the CDRs described herein (e.g., CDRs from any of the anti-PRAME/HLA-A2 antibodies selected from Table 1) so long as immuno specific binding to PRAME/HLA-A2 (e.g., human PRAME/HLA-A2) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% relative to the binding of the original antibody from which it is derived). Any method can be used to ascertain whether immuno specific binding to PRAME/HLA-A2 (e.g., human PRAME/HLA-A2) is maintained, for example, using binding assays and conditions described in the art.
  • PRAME/HLA-A2 e.
  • any of the anti-PRAME/HLA-A2 antibodies of the disclosure have one or more CDR (e.g., HC CDR or LC CDR) sequences substantially similar to any one of the anti-PRAME/HLA-A2 antibodies selected from Table 1.
  • CDR e.g., HC CDR or LC CDR
  • the antibodies may include one or more CDR sequence(s) from any of the anti-PRAME/HLA-A2 antibodies selected from Table 1 containing up to 5, 4, 3, 2, or 1 amino acid residue variations as compared to the corresponding CDR region in any one of the CDRs provided herein (e.g., CDRs from any of the anti-PRAME/HLA-A2 antibodies selected from Table 1) so long as immuno specific binding to PRAME/HLA-A2 (e.g., human PRAME/HLA-A2) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% relative to the binding of the original antibody from which it is derived).
  • PRAME/HLA-A2 e.g., human PRAME/HLA-A2
  • any of the amino acid variations in any of the CDRs provided herein may be conservative variations.
  • Conservative variations can be introduced into the CDRs at positions where the residues are not likely to be involved in interacting with a PRAME/HLA-A2 protein complex (e.g., a human PRAME/HLA-A2 protein complex), for example, as determined based on a crystal structure.
  • PRAME/HLA-A2 protein complex e.g., a human PRAME/HLA-A2 protein complex
  • Some aspects of the disclosure provide anti-PRAME/HLA-A2 antibodies that comprise one or more of the heavy chain variable (VH) and/or light chain variable (VL) domains provided herein.
  • any of the VH domains provided herein include one or more of the HC CDR sequences (e.g., HC CDR1, HC CDR2, and HC CDR3) provided herein, for example, any of the CDR-H sequences provided in any one of the anti-PRAME/HLA-A2 selected from Table 1.
  • any of the VL domains provided herein include one or more of the CDR-L sequences (e.g., LC CDR1, LC CDR2, and LC CDR3) provided herein, for example, any of the LC CDR sequences provided in any one of the anti-PRAME/HLA-A2 antibodies selected from Table 1.
  • the anti-PRAME/HLA-A2 antibodies of the disclosure include any antibody that includes a heavy chain variable domain and/or a light chain variable domain of any one of the anti-PRAME/HLA-A2 antibodies selected from Table 1, and variants thereof.
  • anti-PRAME/HLA-A2 antibodies of the disclosure include any antibody that includes the heavy chain variable and light chain variable pairs of any anti-PRAME/HLA-A2 antibodies selected from Table 1.
  • the anti-PRAME/HLA-A2 antibodies having a heavy chain variable (VH) and/or a light chain variable (VL) domain amino acid sequence homologous to any of those described herein.
  • the anti-PRAME/HLA- A2 antibody comprises a heavy chain variable sequence or a light chain variable sequence that is at least 75% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the heavy chain variable sequence and/or any light chain variable sequence of any one of the anti-PRAME/HLA-A2 antibodies selected from Table 1.
  • the homologous heavy chain variable and/or a light chain variable amino acid sequences do not vary within any of the CDR sequences provided herein.
  • the degree of sequence variation e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%
  • any of the anti-PRAME/HLA-A2 antibodies provided herein comprise a heavy chain variable sequence and a light chain variable sequence that comprises a framework sequence that is at least 75% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the framework sequence of any anti-PRAME/HLA-A2 antibodies selected from Table 1.
  • a framework sequence that is at least 75% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the framework sequence of any anti-PRAME/HLA-A2 antibodies selected from Table 1.
  • the anti-PRAME/HLA-A2 antibody of the present disclosure is a humanized antibody (e.g., a humanized variant containing one or more CDRs of Table 1).
  • the anti-PRAME/HLA-A2 antibody of the present disclosure comprises a HC CDR1, a HC CDR2, a HC CDR3, a LC CDR1, a LC CDR2, and a LC CDR3 that are the same as the HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 shown in Table 1, and comprises a humanized heavy chain variable region and/or a humanized light chain variable region.
  • the anti-PRAME/HLA-A2 antibody of the present disclosure is a humanized antibody comprising a VH containing no more than 20 amino acid variations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) as compared with the VH of any of the anti-PRAME/HLA-A2 antibodies listed in Table 1.
  • a VH containing no more than 20 amino acid variations e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation
  • the anti-PRAME/HLA-A2 antibody of the present disclosure is a humanized antibody comprising a VL containing no more than 20 amino acid variations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) as compared with the VL of any one of the anti- PRAME/HLA-A2 antibodies listed in Table 1.
  • a VH region of the present disclosure comprises HC CDR1 (or a variant thereof in which 1, 2, or 3 amino acids in HC CDR1 are substituted with another amino acid), HC CDR2 (or a variant thereof in which 1, 2, or 3 amino acids in HC CDR2 are substituted with another amino acid) and HC CDR3 (or a variant thereof in which 1, 2, or 3 amino acids in HC CDR3 are substituted with another amino acid) as indicated in: (1) row 1, (2) row 2, (3) row 3, (4) row 4, (5) row 5, (6) row 6, (7) row 7, (8) row 8, (9) row 9, (10) row
  • a VH region of the present disclosure comprises at least 80%, preferably one of ⁇ 80%, ⁇ 85%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, 2?
  • a VL region of the present disclosure comprises LC CDR1 (or a variant thereof in which 1, 2, or 3 amino acids in LC CDR1 are substituted with another amino acid), LC CDR2 (or a variant thereof in which 1, 2, or 3 amino acids in LC CDR2 are substituted with another amino acid) and LC CDR3 (or a variant thereof in which 1, 2, or 3 amino acids in LC CDR3 are substituted with another amino acid) as indicated in: row 1, row 2, row 3, row 4, row 5, row 6, row 7, row 8, row 9, row 10, row 11, row 12, row 13, row 14, row 15, row 16, row 17, row 18, row 19, row 20, row 21, row 22, row 23, row 24, row 25, row 26, row 27, row 28, row 29, row 30, row 31, row 32, and row 33 of Column B of Table 9.
  • a VH region of the present disclosure comprises at least 80%, preferably one of ⁇ 80%, ⁇ 85%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, 2?
  • an antigen-binding molecule according to the present disclosure comprises a VH region according to any one of (1), (2), (3), (4), (5), (6), (7), (8), (9), (10), (11), (12), (13), (14), (15), (16), (17), (18), (19), (20), (21), (22), (23), (24), (25), (26), (27), (28), (29), (30), (31), (32), or (33) as described hereinabove; and a VL region according to any one of (34), (35), (36), (37), (38), (39), (40), (41), (42), (43), (44), (45), (46),
  • an antigen-binding molecule comprises: (1) and (34), (1) and (35), (1) and (36), (1) and (37), (1) and (38), (1) and (39), (1) and (40), (1) and (41), (1) and (42), (1) and (43), (1) and (44), and (1) and (45), (1) and (46), (1) and (47), (1) and (48), (1) and (49), (1) and (50), (1) and (51), (1) and (52), (1) and (53), (1) and (54), (1) and (55), (1) and (56), (1) and (57), (1) and (58), (1) and (59), (1) and (60), (1) and (61), (1) and (62), (1) and (63), (1) and (64), (1) and (65), and (1) and (66), (2) and (34), (2) and (35), (2) and (36), (2) and (37), (2) and (38), (2) and (39), (2) and (40), (2) and (41), (2) and (42), (2) and (43), (2) and (44), and (2) and (45), (2) and (
  • an anti-PRAME/HLA-A2 antibody of the present disclosure comprises a HC CDR1, HC CDR2 and HC CDR3 of a heavy chain variable domain having the amino acid sequence of antibody #1 in row 34; antibody #2 in row 35, antibody #3 in row 36, antibody #4 in row 37, antibody #5 in row 38, antibody #6 in row 39, antibody #7 in row 40, antibody #8 in row 41, antibody #9 in row 42, antibody #10 in row 43, antibody #11 in row 44, antibody #12 in row 45, antibody #13 in row 46, antibody #14 in row 47, antibody #15 in row 48, antibody #16 in row 49, antibody #17 in row 50, antibody #18 in row 51, antibody #19 in row 52, antibody #20 in row 53, antibody #21 in row 54, antibody #22 in row 55, antibody #23 in row 56, antibody #24 in row 57, antibody #25 in for 58, antibody #26 in row 59, antibody #27 in row 60, antibody #28 in row 61, antibody #
  • an anti-PRAME/HLA-A2 antibody of the present disclosure comprises a LC CDR1, LC CDR2 and LC CDR3 of antibody #1 in row 34; antibody #2 in row 35, antibody #3 in row 36, antibody #4 in row 37, antibody #5 in row 38, antibody #6 in row 39, antibody #7 in row 40, antibody #8 in row 41, antibody #9 in row 42, antibody #10 in row 43, antibody #11 in row 44, antibody #12 in row 45, antibody #13 in row 46, antibody #14 in row 47, antibody #15 in row 48, antibody #16 in row 49, antibody #17 in row 50, antibody #18 in row 51, antibody #19 in row 52, antibody #20 in row 53, antibody #21 in row 54, antibody #22 in row 55, antibody #23 in row 56, antibody #24 in row 57, antibody #25 in for 58, antibody #26 in row 59, antibody #27 in row 60, antibody #28 in row 61, antibody #29 in row 62, antibody #30 in row 63, antibody
  • an anti-PRAME/HLA-A2 antibody of the present disclosure comprises a HC CDR1, a HC CDR2, a HC CDR3, a LC CDR1, a LC CDR2, and a LC CDR3 having the amino acid sequences of antibody #1 in row 1, antibody #2 in row 2, antibody #3 in row 3, antibody #4 in row 4, antibody #5 in row 5, antibody #6 in row 6, antibody #7 in row 7, antibody #8 in row 8, antibody #9 in row 9, antibody #10 in row 10, antibody #11 in row 11, antibody #12 in row 12, antibody #13 in row 13, antibody #14 in row 14, antibody, antibody #15 in row 15, antibody #16 in row 16, antibody #17 in row 17, antibody #18 in row
  • an anti-PRAME/HLA-A2 antibody of the present disclosure comprises a HC CDR1, a HC CDR2, and a HC CDR3, which collectively contains no more than 5 amino acid variations (e.g., no more than 5, 4, 3, 2, or 1 amino acid variation) as compared with the HC CDR1, the HC CDR2, and the HC CDR3 amino acid sequences of antibody #1 in row 1, antibody #2 in row 2, antibody #3 in row 3, antibody #4 in row 4, antibody #5 in row 5, antibody #6 in row 6, antibody #7 in row 7, antibody #8 in row 8, antibody #9 in row 9, antibody #10 in row 10, antibody #11 in row 11, antibody #12 in row 12, antibody #13 in row 13, antibody #14 in row 14, antibody, antibody #15 in row 15, antibody #16 in row 16, antibody #17 in row 17, antibody #18 in row 18, antibody #19 in row
  • the anti-PRAME/HLA-A2 antibody of the present disclosure comprises a LC CDR1, a LC CDR2, and a LC CDR3, which collectively contains no more than 5 amino acid variations (e.g., no more than 5, 4, 3, 2 or 1 amino acid variation) as compared with the LC CDR1, the LC CDR2, and the LC CDR3 amino acid sequences of antibody #1 in row 1, antibody #2 in row 2, antibody #3 in row 3, antibody #4 in row 4, antibody #5 in row 5, antibody #6 in row 6, antibody #7 in row 7, antibody #8 in row 8, antibody #9 in row 9, antibody #10 in row 10, antibody #11 in row 11, antibody #12 in row 12, antibody #13 in row 13, antibody #14 in row 14, antibody, antibody #15 in row 15, antibody #16 in row 16, antibody #17 in row 17, antibody #18 in row 18, antibody #19 in row
  • an anti-PRAME/HLA-A2 antibody of the present disclosure comprises a HC CDR1, a HC CDR2, and a HC CDR3 that collectively are at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the HC CDR1, the HC CDR2, and the HC CDR3 amino acid sequences of antibody #1 in row 1, antibody #2 in row 2, antibody #3 in row 3, antibody #4 in row 4, antibody #5 in row 5, antibody #6 in row 6, antibody #7 in row 7, antibody #8 in row 8, antibody #9 in row 9, antibody #10 in row 10, antibody #11 in row 11, antibody #12 in row 12, antibody #13 in row
  • antibody #14 in row 14 antibody, antibody #15 in row 15, antibody #16 in row 16, antibody #17 in row 17, antibody #18 in row 18, antibody #19 in row 19, antibody #20 in row
  • the anti-PRAME/HLA-A2 antibody of the present disclosure comprises a LC CDR1, a LC CDR2, and a LC CDR3 that collectively are at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the LC CDR1, the LC CDR2, and the LC CDR3 amino acid sequences of antibody #1 in row 1, antibody #2 in row 2, antibody #3 in row 3, antibody #4 in row 4, antibody #5 in row 5, antibody #6 in row 6, antibody #7 in row 7, antibody #8 in row 8, antibody #9 in row 9, antibody #10 in row 10, antibody #11 in row 11, antibody #12 in row 12, antibody #13 in row 13, antibody #14 in row
  • antibody #15 in row 15, antibody #16 in row 16, antibody #17 in row 17, antibody #18 in row 18, antibody #19 in row 19, antibody #20 in row 20, antibody #21 in row
  • an anti-PRAME/HLA-A2 antibody of the present disclosure comprises: a HC CDR1 having no more than 3 amino acid variations (e.g., no more than 3, 2, or 1 amino acid variation), a HC CDR2 having no more than 3 amino acid variations (e.g., no more than 3, 2, or 1 amino acid variation), and/or a HC CDR3 having no more than 3 amino acid variations (e.g., no more than 3, 2, or 1 amino acid variation) as compared with the HC CDR1, the HC CDR2, and the HC CDR3 amino acid sequences of antibody #1 in row 1, antibody #2 in row 2, antibody #3 in row 3, antibody #4 in row 4, antibody #5 in row 5, antibody #6 in row 6, antibody #7 in row 7, antibody #8 in row 8, antibody #9 in row 9, antibody #10 in row 10, antibody #11 in row 11, antibody #12 in row 12, antibody #13 in row
  • antibody #14 in row 14 antibody, antibody #15 in row 15, antibody #16 in row 16, antibody #17 in row 17, antibody #18 in row 18, antibody #19 in row 19, antibody #20 in row
  • the anti-PRAME/HLA-A2 antibody of the present disclosure comprises: a LC CDR1 having no more than 3 amino acid variations (e.g., no more than 3, 2, or 1 amino acid variation) a LC CDR2 having no more than 3 amino acid variations (e.g., no more than 3, 2, or 1 amino acid variation), and/or a LC CDR3 having no more than 3 amino acid variations (e.g., no more than 3, 2, or 1 amino acid variation) as compared with the LC CDR1, the LC CDR2, and the LC CDR3 amino acid sequences of antibody #1 in row 1, antibody #2 in row 2, antibody #3 in row 3, antibody #4 in row 4, antibody #5 in row 5, antibody #6 in row 6, antibody #7 in row 7, antibody #8 in row 8, antibody #9 in row 9, antibody #10 in row 10, antibody #11 in row 11, antibody #12 in row 12, antibody #13 in row 13, antibody #14 in row
  • antibody #15 in row 15, antibody #16 in row 16, antibody #17 in row 17, antibody #18 in row 18, antibody #19 in row 19, antibody #20 in row 20, antibody #21 in row
  • an anti-PRAME/HLA-A2 antibody of the present disclosure comprises: a HC CDR1 that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the HC CDR1 a HC CDR2 that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the HC CDR2 and/or a HC CDR3 that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%
  • the anti-PRAME/HLA-A2 antibody of the present disclosure comprises: a LC CDR1 that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the LC CDR1; a LC CDR2 that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the LC CDR2; and/or a LC CDR3 that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%,
  • an anti-PRAME/HLA-A2 antibody of the present disclosure comprises a VH comprising the amino acid sequence of antibody #1 in row 34; antibody #2 in row 35, antibody #3 in row 36, antibody #4 in row 37, antibody #5 in row 38, antibody #6 in row 39, antibody #7 in row 40, antibody #8 in row 41, antibody #9 in row 42, antibody #10 in row 43, antibody #11 in row 44, antibody #12 in row 45, antibody #13 in row 46, antibody #14 in row 47, antibody #15 in row 48, antibody #16 in row 49, antibody #17 in row 50, antibody #18 in row 51, antibody #19 in row 52, antibody #20 in row 53, antibody #21 in row 54, antibody #22 in row 55, antibody #23 in row 56, antibody #24 in row 57, antibody #25 in for 58, antibody #26 in row 59, antibody #27 in row 60, antibody #28 in row 61, antibody #29 in row 62, antibody #30 in row 63, antibody #31 in row 64
  • the anti- PRAME/HLA-A2 antibody of the present disclosure comprises a VL comprising the amino acid sequence of antibody #1 in row 34; antibody #2 in row 35, antibody #3 in row 36, antibody #4 in row 37, antibody #5 in row 38, antibody #6 in row 39, antibody #7 in row 40, antibody #8 in row 41, antibody #9 in row 42, antibody #10 in row 43, antibody #11 in row 44, antibody #12 in row 45, antibody #13 in row 46, antibody #14 in row 47, antibody #15 in row 48, antibody #16 in row 49, antibody #17 in row 50, antibody #18 in row 51, antibody #19 in row 52, antibody #20 in row 53, antibody #21 in row 54, antibody #22 in row 55, antibody #23 in row 56, antibody #24 in row 57, antibody #25 in for 58, antibody #26 in row 59, antibody #27 in row 60, antibody #28 in row 61, antibody #29 in row 62, antibody #30 in row 63, antibody #31 in row 64, antibody #
  • the anti-PRAME/HLA-A2 antibody of the present disclosure comprises a VH containing no more than 20 amino acid variations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) as compared with the VH amino acid sequence of antibody #1 in row 34; antibody #2 in row 35, antibody #3 in row 36, antibody #4 in row 37, antibody #5 in row 38, antibody #6 in row 39, antibody #7 in row 40, antibody #8 in row 41, antibody #9 in row 42, antibody #10 in row 43, antibody #11 in row 44, antibody #12 in row 45, antibody #13 in row 46, antibody #14 in row 47, antibody #15 in row 48, antibody #16 in row 49, antibody #17 in row 50, antibody #18 in row 51, antibody #19 in row 52, antibody #20 in row 53, antibody #21 in row 54, antibody #22 in row 55, antibody #23 in row 56, antibody #24 in row 57, antibody #25 in for
  • the anti-PRAME/HLA-A2 antibody of the present disclosure comprises a VL containing no more than 20 amino acid variations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) as compared with the VL amino acid sequence of antibody #1 in row 34; antibody #2 in row 35, antibody #3 in row 36, antibody #4 in row 37, antibody #5 in row 38, antibody #6 in row 39, antibody #7 in row 40, antibody #8 in row 41, antibody #9 in row 42, antibody #10 in row 43, antibody #11 in row 44, antibody #12 in row 45, antibody #13 in row 46, antibody #14 in row 47, antibody #15 in row 48, antibody #16 in row 49, antibody #17 in row 50, antibody #18 in row 51, antibody #19 in row 52, antibody #20 in row 53, antibody #21 in row 54, antibody #22 in row 55, antibody #23 in row 56, antibody #24 in row 57, antibody #25 in for 58, antibody #
  • the number of amino acid variations may occur within a VH amino acid sequence of antibody #1 in row 34; antibody #2 in row 35, antibody #3 in row 36, antibody #4 in row 37, antibody #5 in row 38, antibody #6 in row 39, antibody #7 in row 40, antibody #8 in row 41, antibody #9 in row 42, antibody #10 in row 43, antibody #11 in row 44, antibody #12 in row 45, antibody #13 in row 46, antibody #14 in row 47, antibody #15 in row 48, antibody #16 in row 49, antibody #17 in row 50, antibody #18 in row 51, antibody #19 in row 52, antibody #20 in row 53, antibody #21 in row 54, antibody #22 in row 55, antibody #23 in row 56, antibody #24 in row 57, antibody #25 in for 58, antibody #26 in row 59, antibody #27 in row 60, antibody #28 in row 61, antibody #29
  • an anti-PRAME/HLA-A2 antibodies provided herein comprise a heavy chain variable sequence that comprises a framework sequence that contains no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation to the framework sequence of a VH amino acid sequence of antibody #1 in row 34; antibody #2 in row 35, antibody #3 in row 36, antibody #4 in row 37, antibody #5 in row 38, antibody #6 in row 39, antibody #7 in row 40, antibody #8 in row 41, antibody #9 in row 42, antibody #10 in row 43, antibody #11 in row 44, antibody #12 in row 45, antibody #13 in row 46, antibody #14 in row 47, antibody #15 in row 48, antibody #16 in row 49, antibody #17 in row 50, antibody #18 in row 51, antibody #19 in row 52, antibody #20 in row 53, antibody #21 in row
  • a light chain variable sequence that comprises a framework sequence that that contains no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation to the framework sequence of a VL amino acid sequence of antibody #1 in row 34; antibody #2 in row 35, antibody #3 in row 36, antibody #4 in row 37, antibody #5 in row 38, antibody #6 in row 39, antibody #7 in row 40, antibody #8 in row 41, antibody #9 in row 42, antibody #10 in row 43, antibody #11 in row 44, antibody #12 in row 45, antibody #13 in row 46, antibody #14 in row 47, antibody #15 in
  • the anti-PRAME/HLA-A2 antibody of the present disclosure comprises a VH comprising an amino acid sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VH amino acid sequence of antibody #1 in row 34; antibody #2 in row 35, antibody #3 in row 36, antibody #4 in row 37, antibody #5 in row 38, antibody #6 in row 39, antibody #7 in row 40, antibody #8 in row 41, antibody #9 in row 42, antibody #10 in row 43, antibody #11 in row 44, antibody #12 in row 45, antibody #13 in row 46, antibody #14 in row 47, antibody #15 in row 48, antibody #16 in row 49, antibody #17 in row 50, antibody #18 in row 51, antibody #19 in row 52, antibody #20 in row 53,
  • the anti-PRAME/HLA-A2 antibody of the present disclosure comprises a VL comprising an amino acid sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VL antibody #1 in row 34; antibody #2 in row 35, antibody #3 in row 36, antibody #4 in row 37, antibody #5 in row 38, antibody #6 in row 39, antibody #7 in row 40, antibody #8 in row 41, antibody #9 in row 42, antibody #10 in row 43, antibody #11 in row 44, antibody #12 in row
  • the degree of sequence variation (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) may occur within a VH of antibody #1 in row 34; antibody #2 in row 35, antibody #3 in row 36, antibody #4 in row 37, antibody #5 in row 38, antibody #6 in row 39, antibody #7 in row 40, antibody #8 in row 41, antibody #9 in row 42, antibody #10 in row 43, antibody #11 in row 44, antibody #12 in row 45, antibody #13 in row
  • antibody #29 in row 62 antibody #30 in row 63, antibody #31 in row 64, antibody #32 in row 65, or antibody #33 in row 66, column A of Table 10, and/or a VL of antibody #1 in row 34; antibody #2 in row 35, antibody #3 in row 36, antibody #4 in row 37, antibody #5 in row 38, antibody #6 in row 39, antibody #7 in row 40, antibody #8 in row 41, antibody #9 in row 42, antibody #10 in row 43, antibody #11 in row 44, antibody #12 in row 45, antibody #13 in row 46, antibody #14 in row 47, antibody #15 in row 48, antibody #16 in row 49, antibody #17 in row 50, antibody #18 in row 51, antibody #19 in row 52, antibody #20 in row 53, antibody #21 in row 54, antibody #22 in row 55, antibody #23 in row 56, antibody #24 in row 57, antibody #25 in for 58, antibody #26 in row 59, antibody #27 in row 60, antibody #28 in row 61, antibody #29 in
  • an anti-PRAME/HLA-A2 antibody provided herein comprise a heavy chain variable sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the framework sequence of a VH of antibody #1 in row 34; antibody #2 in row 35, antibody #3 in row 36, antibody #4 in row 37, antibody #5 in row 38, antibody #6 in row 39, antibody #7 in row 40, antibody #8 in row 41, antibody #9 in row 42, antibody #10 in row 43, antibody #11 in row 44, antibody #12 in row 45, antibody #13 in row 46, antibody #14 in row 47, antibody #15 in row 48, antibody #16 in row 49, antibody #17 in row 50, antibody #18 in row 51, antibody #19 in row 52, antibody #20 in row 53, antibody #21 in row 54,
  • the anti-PRAME/HLA-A2 antibody of the present disclosure is a chimeric antibody, which can include a heavy constant region and a light constant region from a human antibody.
  • Chimeric antibodies refer to antibodies having a variable region or part of variable region from a first species and a constant region from a second species.
  • the variable region of both light and heavy chains mimics the variable regions of antibodies derived from one species of mammals (e.g., a non-human mammal such as mouse, rabbit, and rat), while the constant portions are homologous to the sequences in antibodies derived from another mammal such as human.
  • amino acid modifications can be made in the variable region and/or the constant region.
  • the anti-PRAME/HLA-A2 antibody described herein is a chimeric antibody, which can include a heavy constant region and a light constant region from a human antibody.
  • Chimeric antibodies refer to antibodies having a variable region or part of variable region from a first species and a constant region from a second species.
  • the variable region of both light and heavy chains mimics the variable regions of antibodies derived from one species of mammals e.g., a non- human mammal such as mouse, rabbit, and rat), while the constant portions are homologous to the sequences in antibodies derived from another mammal such as human.
  • amino acid modifications can be made in the variable region and/or the constant region.
  • the anti-PRAME/HLA-A2 antibody of the present disclosure comprises a VL domain and/or VH domain of any one of the anti-PRAME/HLA- A2 antibodies selected from Table 1, and comprises a constant region comprising the amino acid sequences of the constant regions of an IgG, IgE, IgM, IgD, IgA or IgY immunoglobulin molecule, any class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2), or any subclass (e.g., IgG2a and IgG2b) of immunoglobulin molecule.
  • Non-limiting examples of human constant regions are described in the art, e.g., see Kabat E A et al., (1991) supra.
  • the light chain of any of the anti-PRAME/HLA-A2 antibodies described herein may further comprise a light chain constant region (CL), which can be any CL known in the art.
  • CL is a kappa light chain.
  • the CL is a lambda light chain.
  • antibody heavy and light chain constant regions may be used in some embodiments, e.g., those provided in the IMGT database (www.imgt.org) or at www.vbase2.org/vbstat.php., both of which are incorporated by reference herein.
  • the anti-PRAME/HLA-A2 antibodies described herein can be in any antibody form, including, but not limited to, intact (i.e., full-length) antibodies, antigen-binding fragments thereof (such as Fab, F(ab'), F(ab')2, Fv), single chain antibodies, bi-specific antibodies, or nanobodies.
  • the anti-PRAME/HLA-A2 antibody described herein is a scFv.
  • the anti-PRAME/HLA-A2 antibody described herein is a scFv-Fab (e.g., scFv fused to a portion of a constant region).
  • a bispecific antibody is provided herein, in some embodiments, provided herein, in some embodiments, is a bispecific antibody.
  • the first antigen binding site of the bispecific antibody specifically binds PRAME/HLA-A2.
  • the second antigen binding site specifically binds a T cell antigen.
  • the T cell antigen is a CD3 complex or a portion thereof.
  • conservative mutations can be introduced into antibody sequences (e.g., CDRs or framework sequences) at positions where the residues are not likely to be involved in interacting with a target antigen (e.g., PRAME/HLA-A2), for example, as determined based on a crystal structure.
  • a target antigen e.g., PRAME/HLA-A2
  • one, two or more mutations are introduced into the Fc region of an anti-PRAME/HLA-A2 antibody described herein (e.g., in a CH2 domain (residues 231-340 of human IgGl) and/or CH3 domain (residues 341-447 of human IgGl) and/or the hinge region, with numbering according to the Kabat numbering system (e.g., the EU index in Kabat) to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding and/or antigen-dependent cellular cytotoxicity.
  • Kabat numbering system e.g., the EU index in Kabat
  • one, two or more mutations are introduced into the hinge region of the Fc region (CHI domain) such that the number of cysteine residues in the hinge region are altered (e.g., increased or decreased) as described in, e.g., U.S. Pat. No. 5,677,425.
  • the number of cysteine residues in the hinge region of the CHI domain can be altered to, e.g., facilitate assembly of the light and heavy chains, or to alter (e.g., increase or decrease) the stability of an antibody or to facilitate linker conjugation.
  • one, two or more mutations are introduced into the Fc region of an antibody described herein (e.g., in a CH2 domain (residues 231-340 of human IgGl) and/or CH3 domain (residues 341-447 of human IgGl) and/or the hinge region, with numbering according to the Kabat numbering system (e.g., the EU index in Kabat) to increase or decrease the affinity of an antibody for an Fc receptor (e.g., an activated Fc receptor) on the surface of an effector cell.
  • Kabat numbering system e.g., the EU index in Kabat
  • Mutations in the Fc region of an antibody that decrease or increase the affinity of an antibody for an Fc receptor and techniques for introducing such mutations into the Fc receptor or fragment thereof are known to one of skill in the art. Examples of mutations in the Fc receptor of an antibody that can be made to alter the affinity of the antibody for an Fc receptor are described in, e.g., Smith P et al., (2012) PNAS 109: 6181-6186, U.S. Pat. No. 6,737,056, and International Publication Nos. WO 02/060919; WO 98/23289; and WO 97/34631, which are incorporated herein by reference.
  • one, two or more amino acid mutations are introduced into an IgG constant domain, or FcRn-binding fragment thereof (preferably an Fc or hinge-Fc domain fragment) to alter (e.g., decrease or increase) half-life of an antibody in vivo.
  • an IgG constant domain, or FcRn-binding fragment thereof preferably an Fc or hinge-Fc domain fragment
  • one, two or more amino acid mutations are introduced into an IgG constant domain, or FcRn-binding fragment thereof (preferably an Fc or hinge-Fc domain fragment) to decrease the half-life of the anti-PRAME/HLA-A2 antibody in vivo.
  • one, two or more amino acid mutations are introduced into an IgG constant domain, or FcRn-binding fragment thereof (preferably an Fc or hinge-Fc domain fragment) to increase the half-life of an antibody in vivo.
  • the antibodies can have one or more amino acid mutations (e.g., substitutions) in the second constant (CH2) domain (residues 231-340 of human IgGl) and/or the third constant (CH3) domain (residues 341-447 of human IgGl), with numbering according to the EU index in Kabat (Kabat E A et al., (1991) supra).
  • substitutions e.g., substitutions in the second constant (CH2) domain
  • CH3 domain residues 341-447 of human IgGl
  • the constant region of the IgGl of an antibody described herein comprises a methionine (M) to tyrosine (Y) substitution in position 252, a serine (S) to threonine (T) substitution in position 254, and a threonine (T) to glutamic acid (E) substitution in position 256, numbered according to the EU index as in Kabat. See U.S. Pat. No. 7,658,921, which is incorporated herein by reference.
  • an antibody comprises an IgG constant domain comprising one, two, three or more amino acid substitutions of amino acid residues at positions 251-257, 285-290, 308-314, 385-389, and 428-436, numbered according to the EU index as in Kabat.
  • one, two or more amino acid substitutions are introduced into an IgG constant domain Fc region to alter the effector function(s) of the anti- PRAME/HLA-A2 antibody.
  • the effector ligand to which affinity is altered can be, for example, an Fc receptor or the Cl component of complement. This approach is described in further detail in U.S. Pat. Nos. 5,624,821 and 5,648,260 (e.g., L234A and L235A mutations).
  • the deletion or inactivation (through point mutations or other means) of a constant region domain can reduce Fc receptor binding of the circulating antibody thereby increasing tumor localization. See, e.g., U.S. Pat. Nos.
  • one or more amino acid substitutions may be introduced into the Fc region of an antibody described herein to remove potential glycosylation sites on Fc region, which may reduce Fc receptor binding (see, e.g., Shields R L et al., (2001) J Biol Chem 276: 6591-604).
  • one or more amino in the constant region of an anti- PRAME/HLA-A2 antibody described herein can be replaced with a different amino acid residue such that the antibody has altered Clq binding and/or reduced or abolished complement dependent cytotoxicity (CDC).
  • CDC complement dependent cytotoxicity
  • one or more amino acid residues in the N-terminal region of the CH2 domain of an antibody described herein are altered to thereby alter the ability of the antibody to fix complement. This approach is described further in International Publication No. WO 94/29351.
  • the Fc region of an antibody described herein is modified to increase the ability of the antibody to mediate antibody dependent cellular cytotoxicity (ADCC) and/or to increase the affinity of the antibody for an Fey receptor.
  • ADCC antibody dependent cellular cytotoxicity
  • the heavy and/or light chain variable domain(s) sequence(s) of the antibodies provided herein can be used to generate, for example, CDR-grafted, chimeric, humanized, or composite human antibodies or antigen-binding fragments, as described elsewhere herein.
  • any variant, CDR-grafted, chimeric, humanized, or composite antibodies derived from any of the antibodies provided herein may be useful in the compositions and methods described herein and will maintain the ability to specifically bind PRAME/HLA-A2, such that the variant, CDR-grafted, chimeric, humanized, or composite antibody has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or more binding to PRAME/HLA-A2 relative to the original antibody from which it is derived.
  • the antibodies provided herein comprise mutations that confer desirable properties to the antibodies.
  • the antibodies provided herein may comprise a stabilizing ‘Adair’ mutation (Angal S., et al., “A single amino acid substitution abolishes the heterogeneity of chimeric mouse/human (IgG4) antibody,” Mol Immunol 30, 105-108; 1993), where serine 228 (EU numbering; residue 241 Kabat numbering) is converted to proline resulting in an IgGl-like hinge sequence.
  • any of the antibodies may include a stabilizing ‘Adair’ mutation.
  • an antibody is modified, e.g., modified via glycosylation, phosphorylation, sumoylation, and/or methylation.
  • an antibody is a glycosylated antibody, which is conjugated to one or more sugar or carbohydrate molecules.
  • the one or more sugar or carbohydrate molecules are conjugated to an antibody via N-glycosylation, O-glycosylation, C-glycosylation, glypiation (GPI anchor attachment), and/or phosphoglycosylation.
  • the one or more sugar or carbohydrate molecules are monosaccharides, disaccharides, oligosaccharides, or glycans.
  • the one or more sugar or carbohydrate molecule is a branched oligosaccharide or a branched glycan.
  • the one or more sugar or carbohydrate molecule includes a mannose unit, a glucose unit, an N-acetylglucosamine unit, an N-acetylgalactosamine unit, a galactose unit, a fucose unit, or a phospholipid unit.
  • a glycosylated antibody is fully or partially glycosylated.
  • an antibody is glycosylated by chemical reactions or by enzymatic means.
  • an antibody is glycosylated in vitro or inside a cell, which may optionally be deficient in an enzyme in the N- or O- glycosylation pathway, e.g., a glycosyltransferase.
  • an antibody is functionalized with sugar or carbohydrate molecules as described in International Patent Application Publication WO2014065661, published on May 1, 2014, entitled, “Modified antibody, antibodyconjugate and process for the preparation thereof ’ .
  • any one of the anti-PRAME/HLA-A2 antibodies described herein may comprise a signal peptide in the heavy and/or light chain sequence (e.g., a N- terminal signal peptide).
  • the anti-PRAME/HLA-A2 antibody described herein comprises any one of the VH and VL sequences, any one of the IgG heavy chain and light chain sequences, or any one of the F(ab') heavy chain and light chain sequences described herein, and further comprises a signal peptide (e.g., a N-terminal signal peptide).
  • any one of the antibodies described herein is a multispecific antibody that specifically binds PRAME/HLA-A2 and one or more additional target antigens.
  • an antibody is a bispecific antibody that specifically binds to PRAME/HLA-A2 and one additional target antigen.
  • the multispecific antibody or bispecific antibody can be obtained by known technology in the art.
  • the one or more additional targets include but are not limited to CD3, CD4, CD8, CD20, CD19, CD21, CD23, CD46, CD80, HLA-DR, CD74, CD22, CD14, CD15, CD16, CD123, TCR gamma/delta, NKp46, or KIR.
  • the antibodies described herein are conjugated directly or indirectly to one or more molecular pay loads or labels.
  • antibodies described herein are conjugated to molecular payload, e.g., a molecular payload providing a therapeutic benefit for a subject, e.g., an antibody-drug conjugate (ADC).
  • ADC antibody-drug conjugate
  • a molecular payload may be a small molecule, protein, nucleic acid, oligonucleotide, or any molecular entity capable of modulating the activity or function of a gene, protein, and/or nucleic acid, e.g., in a cell.
  • the molecular pay load is a cytotoxic agent or a chemotherapeutic agent.
  • antibodies described herein are conjugated directly or indirectly to a detectable label, e.g., for diagnostic purposes.
  • the present disclosure also provides fusion proteins comprising an anti-PRAME/HLA-A2 antibody described herein fused directly or indirectly (e.g., via a linker) to one or more polypeptide or protein.
  • the present disclosure also contemplates engineering any of the anti-PRAME/HLA-A2 antibodies or antigen binding fragment thereof disclosed herein into the format of an extracellular ligand-binding domain of a CAR expressed by genetically modified immune cells (e.g., T cells, NK cells, or NKT cells) described herein.
  • Aspects of the present disclosure also provide chimeric antigen receptors (CARs) comprising an extracellular ligand-binding domain.
  • the ligand-binding domain binds to a cell surface marker on a target cell (e.g., a tumor cell or cancer cell).
  • the ligand-binding domain binds to a cell surface marker on a target cell in a particular disease state (e.g., PRAME/HLA-A2 on a PRAME-expressing tumor cell or cancer cell).
  • a CAR e.g., anti-PRAME/HLA-A2 CAR
  • the extracellular domain of an anti-PRAME/HLA-A2 CAR comprises a target- specific binding element (e.g., an antibody that specifically binds to PRAME/HLA-A2 (e.g., human PRAME/HLA-A2)) otherwise referred to herein as a ligand-binding domain (also referred to herein as an antigen-binding domain).
  • a target-specific binding element e.g., an antibody that specifically binds to PRAME/HLA-A2 (e.g., human PRAME/HLA-A2)
  • a ligand-binding domain also referred to herein as an antigen-binding domain.
  • the extracellular ligand-binding domain of an anti-PRAME/HLA-A2 CAR is an antigen-binding domain or a portion thereof.
  • the extracellular ligand-binding domain of an anti-PRAME/HLA-A2 CAR is a Fab.
  • the extracellular ligand-binding domain of an anti- PRAME/HLA-A2 CAR is a scFv.
  • an extracellular ligand-binding domain of a CAR described herein comprises an anti-PRAME/HLA-A2 antibody as described in Table 1.
  • an anti-PRAME/HLA-A2 CAR of the present disclosure comprises a HC CDR1, HC CDR2 and HC CDR3 of a heavy chain variable domain having the amino acid sequence of antibody #1 in row 34; antibody #2 in row 35, antibody #3 in row 36, antibody #4 in row 37, antibody #5 in row 38, antibody #6 in row 39, antibody #7 in row 40, antibody #8 in row 41, antibody #9 in row 42, antibody #10 in row 43, antibody #11 in row 44, antibody #12 in row 45, antibody #13 in row 46, antibody #14 in row 47, antibody #15 in row 48, antibody #16 in row 49, antibody #17 in row 50, antibody #18 in row 51, antibody #19 in row 52, antibody #20 in row 53, antibody #21 in row 54, antibody #22 in row 55, antibody #23 in row 56, antibody #24 in row 57, antibody #25 in for 58, antibody #26 in row 59, antibody #27 in row 60, antibody #28 in row 61, antibody
  • an anti-PRAME/HLA-A2 CAR of the present disclosure comprises a LC CDR1, LC CDR2 and LC CDR3 of a light chain variable domain having the amino acid sequence of antibody #1 in row 34; antibody #2 in row 35, antibody #3 in row 36, antibody #4 in row 37, antibody #5 in row 38, antibody #6 in row 39, antibody #7 in row 40, antibody #8 in row 41, antibody #9 in row 42, antibody #10 in row 43, antibody #11 in row 44, antibody #12 in row 45, antibody #13 in row 46, antibody #14 in row 47, antibody #15 in row 48, antibody #16 in row 49, antibody #17 in row 50, antibody #18 in row 51, antibody #19 in row 52, antibody #20 in row 53, antibody #21 in row 54, antibody #22 in row 55, antibody #23 in row 56, antibody #24 in row 57, antibody #25 in for 58, antibody #26 in row 59, antibody #27 in row 60, antibody #28 in row 61, antibody #29 in
  • an anti-PRAME/HLA-A2 CAR of the present disclosure comprises a HC CDR1, a HC CDR2, a HC CDR3, a LC CDR1, a LC CDR2, a LC CDR3 having the amino acid sequences of antibody #1 in row 1, antibody #2 in row 2, antibody #3 in row 3, antibody #4 in row 4, antibody #5 in row 5, antibody #6 in row 6, antibody #7 in row 7, antibody #8 in row 8, antibody #9 in row 9, antibody #10 in row 10, antibody #11 in row 11, antibody #12 in row 12, antibody #13 in row 13, antibody #14 in row 14, antibody, antibody #15 in row 15, antibody #16 in row 16, antibody #17 in row 17, antibody #18 in row
  • an anti-PRAME/HLA-A2 CAR of the present disclosure comprises a HC CDR1, a HC CDR2, and a HC CDR3, which collectively contains no more than 5 amino acid variations (e.g., no more than 5, 4, 3, 2, or 1 amino acid variation) as compared with the HC CDR1, the HC CDR2, and the HC CDR3 amino acid sequences of antibody #1 in row 1, antibody #2 in row 2, antibody #3 in row 3, antibody #4 in row 4, antibody #5 in row 5, antibody #6 in row 6, antibody #7 in row 7, antibody #8 in row 8, antibody #9 in row 9, antibody #10 in row 10, antibody #11 in row 11, antibody #12 in row 12, antibody #13 in row 13, antibody #14 in row 14, antibody, antibody #15 in row 15, antibody #16 in row 16, antibody #17 in row 17, antibody #18 in row 18, antibody #19 in row
  • an anti-PRAME/HLA-A2 CAR of the present disclosure comprises a LC CDR1, a LC CDR2, and a LC CDR3, which collectively contains no more than 5 amino acid variations (e.g., no more than 5, 4, 3, 2 or 1 amino acid variation) as compared with the LC CDR1, the LC CDR2, and the LC CDR3 amino acid sequences of antibody #1 in row 1, antibody #2 in row 2, antibody #3 in row 3, antibody #4 in row 4, antibody #5 in row 5, antibody #6 in row 6, antibody #7 in row 7, antibody #8 in row 8, antibody #9 in row 9, antibody #10 in row 10, antibody #11 in row 11, antibody #12 in row 12, antibody #13 in row 13, antibody #14 in row 14, antibody, antibody #15 in row 15, antibody #16 in row 16, antibody #17 in row 17, antibody #18 in row 18, antibody #19 in row 19, antibody #20 in row 20, antibody #21 in row 21, antibody #22 in row 22, antibody #23 in row 23, antibody #24 in row 24, antibody #25 in row 25, antibody #26
  • an anti-PRAME/HLA-A2 CAR of the present disclosure comprises a HC CDR1, a HC CDR2, and a HC CDR3 that collectively are at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the HC CDR1, the HC CDR2, and the HC CDR3 amino acid sequences of antibody #1 in row 1, antibody #2 in row 2, antibody #3 in row 3, antibody #4 in row 4, antibody #5 in row 5, antibody #6 in row 6, antibody #7 in row 7, antibody #8 in row 8, antibody #9 in row 9, antibody #10 in row 10, antibody #11 in row 11, antibody #12 in row 12, antibody #13 in row
  • antibody #14 in row 14 antibody, antibody #15 in row 15, antibody #16 in row 16, antibody #17 in row 17, antibody #18 in row 18, antibody #19 in row 19, antibody #20 in row
  • an anti-PRAME/HLA-A2 CAR of the present disclosure comprises a LC CDR1, a LC CDR2, and a LC CDR3 that collectively are at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the to the LC CDR1, the LC CDR2, and the LC CDR3 amino acid sequences of antibody #1 in row 1, antibody #2 in row 2, antibody #3 in row 3, antibody #4 in row 4, antibody #5 in row 5, antibody #6 in row 6, antibody #7 in row 7, antibody #8 in row 8, antibody #9 in row 9, antibody #10 in row 10, antibody #11 in row 11, antibody #12 in row 12, antibody #13 in row 13, antibody #14 in row
  • antibody #15 in row 15, antibody #16 in row 16, antibody #17 in row 17, antibody #18 in row 18, antibody #19 in row 19, antibody #20 in row 20, antibody #21 in row
  • an anti-PRAME/HLA-A2 CAR of the present disclosure comprises: a HC CDR1 having no more than 3 amino acid variations (e.g., no more than 3, 2, or 1 amino acid variation); a HC CDR2 having no more than 3 amino acid variations (e.g., no more than 3, 2, or 1 amino acid variation); and/or a HC CDR3 having no more than 3 amino acid variations (e.g., no more than 3, 2, or 1 amino acid variation) as compared with the HC CDR1, the HC CDR2, and the HC CDR3 amino acid sequences of antibody #1 in row 1, antibody #2 in row 2, antibody #3 in row 3, antibody #4 in row 4, antibody #5 in row 5, antibody #6 in row 6, antibody #7 in row 7, antibody #8 in row 8, antibody #9 in row 9, antibody #10 in row 10, antibody #11 in row 11, antibody #12 in row 12, antibody #13 in row
  • antibody #14 in row 14 antibody, antibody #15 in row 15, antibody #16 in row 16, antibody #17 in row 17, antibody #18 in row 18, antibody #19 in row 19, antibody #20 in row
  • an anti-PRAME/HLA-A2 CAR of the present disclosure comprises: a LC CDR1 having no more than 3 amino acid variations (e.g., no more than 3, 2, or 1 amino acid variation); a LC CDR2 having no more than 3 amino acid variations (e.g., no more than 3, 2, or 1 amino acid variation); and/or a LC CDR3 having no more than 3 amino acid variations (e.g., no more than 3, 2, or 1 amino acid variation) as compared with the LC CDR1, the LC CDR2, and the LC CDR3 amino acid sequences of antibody #1 in row 1, antibody #2 in row 2, antibody #3 in row 3, antibody #4 in row 4, antibody #5 in row 5, antibody #6 in row 6, antibody #7 in row 7, antibody #8 in row 8, antibody #9 in row 9, antibody #10 in row 10, antibody #11 in row 11, antibody #12 in row 12, antibody #13 in row 13, antibody #14 in row
  • antibody #15 in row 15, antibody #16 in row 16, antibody #17 in row 17, antibody #18 in row 18, antibody #19 in row 19, antibody #20 in row 20, antibody #21 in row
  • an anti-PRAME/HLA-A2 CAR of the present disclosure comprises: a HC CDR1 that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the HC CDR1; a HC CDR2 that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the HC CDR2; and/or a HC CDR3 that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%,
  • an anti-PRAME/HLA-A2 CAR of the present disclosure comprises: a LC CDR1 that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the LC CDR1; a LC CDR2 that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the LC CDR2; and/or a LC CDR3 that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%
  • an anti-PRAME/HLA-A2 CAR of the present disclosure comprises a VH comprising the amino acid sequence of antibody #1 in row 34; antibody #2 in row 35, antibody #3 in row 36, antibody #4 in row 37, antibody #5 in row 38, antibody #6 in row 39, antibody #7 in row 40, antibody #8 in row 41, antibody #9 in row 42, antibody #10 in row 43, antibody #11 in row 44, antibody #12 in row 45, antibody #13 in row 46, antibody #14 in row 47, antibody #15 in row 48, antibody #16 in row 49, antibody #17 in row 50, antibody #18 in row 51, antibody #19 in row 52, antibody #20 in row 53, antibody #21 in row 54, antibody #22 in row 55, antibody #23 in row 56, antibody #24 in row 57, antibody #25 in for 58, antibody #26 in row 59, antibody #27 in row 60, antibody #28 in row 61, antibody #29 in row 62, antibody #30 in row 63, antibody #31 in
  • an anti- PRAME/HLA-A2 CAR of the present disclosure comprises a VL comprising the amino acid sequence of antibody #1 in row 34; antibody #2 in row 35, antibody #3 in row 36, antibody #4 in row 37, antibody #5 in row 38, antibody #6 in row 39, antibody #7 in row 40, antibody #8 in row 41, antibody #9 in row 42, antibody #10 in row 43, antibody #11 in row 44, antibody #12 in row 45, antibody #13 in row 46, antibody #14 in row 47, antibody #15 in row 48, antibody #16 in row 49, antibody #17 in row 50, antibody #18 in row 51, antibody #19 in row 52, antibody #20 in row 53, antibody #21 in row 54, antibody #22 in row 55, antibody #23 in row 56, antibody #24 in row 57, antibody #25 in for 58, antibody #26 in row 59, antibody #27 in row 60, antibody #28 in row 61, antibody #29 in row 62, antibody #30 in row 63, antibody #31 in row 64, antibody
  • an anti-PRAME/HLA-A2 CAR of the present disclosure comprises a VH containing no more than 20 amino acid variations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) as compared with the VH amino acid sequence of antibody #1 in row 34; antibody #2 in row 35, antibody #3 in row 36, antibody #4 in row 37, antibody #5 in row 38, antibody #6 in row 39, antibody #7 in row 40, antibody #8 in row 41, antibody #9 in row 42, antibody #10 in row 43, antibody #11 in row 44, antibody #12 in row 45, antibody #13 in row 46, antibody #14 in row 47, antibody #15 in row 48, antibody #16 in row 49, antibody #17 in row 50, antibody #18 in row 51, antibody #19 in row 52, antibody #20 in row 53, antibody #21 in row 54, antibody #22 in row 55, antibody #23 in row 56, antibody #24 in row 57, antibody #25 in for
  • an anti-PRAME/HLA-A2 CAR of the present disclosure comprises a VL containing no more than 20 amino acid variations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) as compared with the VL amino acid sequence of antibody #1 in row 34; antibody #2 in row 35, antibody #3 in row 36, antibody #4 in row 37, antibody #5 in row 38, antibody #6 in row 39, antibody #7 in row 40, antibody #8 in row 41, antibody #9 in row 42, antibody #10 in row 43, antibody #11 in row 44, antibody #12 in row 45, antibody #13 in row 46, antibody #14 in row 47, antibody #15 in row 48, antibody #16 in row 49, antibody #17 in row 50, antibody #18 in row 51, antibody #19 in row 52, antibody #20 in row 53, antibody #21 in row 54, antibody #22 in row 55, antibody #23 in row 56, antibody #24 in row 57, antibody #25 in for 58, antibody
  • the number of amino acid variations may occur within a VH of amino acid sequence of antibody #1 in row 34; antibody #2 in row 35, antibody #3 in row 36, antibody #4 in row 37, antibody #5 in row 38, antibody #6 in row 39, antibody #7 in row 40, antibody #8 in row 41, antibody #9 in row 42, antibody #10 in row 43, antibody #11 in row 44, antibody #12 in row 45, antibody #13 in row 46, antibody #14 in row 47, antibody #15 in row 48, antibody #16 in row 49, antibody #17 in row 50, antibody #18 in row 51, antibody #19 in row 52, antibody #20 in row 53, antibody #21 in row 54, antibody #22 in row 55, antibody #23 in row 56, antibody #24 in row 57, antibody #25 in for 58, antibody #26 in row 59, antibody #27 in row 60, antibody #28 in row 61, antibody #
  • an anti-PRAME/HLA-A2 CAR comprise a heavy chain variable sequence that comprises a framework sequence that contains no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation to the framework sequence of a VH amino acid sequence of antibody #1 in row 34; antibody #2 in row 35, antibody #3 in row 36, antibody #4 in row 37, antibody #5 in row 38, antibody #6 in row 39, antibody #7 in row 40, antibody #8 in row 41, antibody #9 in row 42, antibody #10 in row 43, antibody #11 in row 44, antibody #12 in row 45, antibody #13 in row 46, antibody #14 in row 47, antibody #15 in row 48, antibody #16 in row 49, antibody #17 in row 50, antibody #18 in row 51, antibody #19 in row 52, antibody #20 in row 53, antibody #21 in row 54, antibody #22 in row 55, antibody #23 in row 56, antibody #24 in row 57, antibody #25 in for 58, antibody #26 in row
  • an anti-PRAME/HLA-A2 CAR of the present disclosure comprises a VH comprising an amino acid sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VH as set forth in amino acid sequence of antibody #1 in row 34; antibody #2 in row 35, antibody #3 in row 36, antibody #4 in row 37, antibody #5 in row 38, antibody #6 in row 39, antibody #7 in row 40, antibody #8 in row 41, antibody #9 in row 42, antibody #10 in row 43, antibody #11 in row 44, antibody #12 in row 45, antibody #13 in row 46, antibody #14 in row 47, antibody #15 in row 48, antibody #16 in row 49, antibody #17 in row 50, antibody #18 in row 51, antibody #19 in row 52, antibody #
  • an anti-PRAME/HLA-A2 CAR of the present disclosure comprises a VL comprising an amino acid sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VL as set forth in amino acid sequence of antibody #1 in row 34; antibody #2 in row 35, antibody #3 in row 36, antibody #4 in row 37, antibody #5 in row 38, antibody #6 in row 39, antibody #7 in row 40, antibody #8 in row 41, antibody #9 in row 42, antibody #10 in row 43, antibody #11 in row 44, antibody #12 in row 45, antibody #13 in row 46, antibody #14 in row 47, antibody #15 in row 48, antibody #16 in row 49, antibody #17 in row 50, antibody #18 in row 51, antibody #19 in row 52, antibody #20 in row
  • the degree of sequence variation (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) may occur within a VH of amino acid sequence of antibody #1 in row 34; antibody #2 in row 35, antibody #3 in row 36, antibody #4 in row 37, antibody #5 in row 38, antibody #6 in row 39, antibody #7 in row 40, antibody #8 in row 41, antibody #9 in row 42, antibody #10 in row 43, antibody #11 in row 44, antibody #12 in row 45, antibody #13 in row 46, antibody #14 in row 47, antibody #15 in row 48, antibody #16 in row 49, antibody #17 in row 50, antibody #18 in row 51, antibody #19 in row 52, antibody #20 in row 53, antibody #21 in row 54, antibody #22 in row 55, antibody #23 in row 56, antibody #24 in row 57,
  • an anti-PRAME/HLA-A2 CAR comprise a heavy chain variable sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the framework sequence of a VH of amino acid sequence of antibody #1 in row 34; antibody #2 in row 35, antibody #3 in row 36, antibody #4 in row 37, antibody #5 in row 38, antibody #6 in row 39, antibody #7 in row 40, antibody #8 in row 41, antibody #9 in row 42, antibody #10 in row 43, antibody #11 in row 44, antibody #12 in row 45, antibody #13 in row 46, antibody #14 in row 47, antibody #15 in row 48, antibody #16 in row 49, antibody #17 in row 50, antibody #18 in row 51, antibody #19 in row 52, antibody #20 in row 53, antibody #
  • a chimeric antigen receptor (CAR) of the present disclosure comprises a HC CDR1 having the amino acid sequence of X1X2X3X4X5WX6, a HC CDR2 having the amino acid sequence of SEQ ID NO: 2, a HC CDR3 having the amino acid sequence of X11X12X13X14IRGX15X16X17X18X19, a LC CDR1 having the amino acid sequence of SEQ ID NO: 4, a LC CDR2 having the amino acid sequence of SEQ ID NO: 5, and a LC CDR3 having the amino acid sequence of SEQ ID NO: 6.
  • a CAR comprises a HC CDR1 having the amino acid sequence of X1X2X3X4X5WX6, wherein Xi is S or N; X2 is Y, N, or G; X3 is F, N or S; X4 is H or absent; X5 is W, Y or absent; and Xf> is N or S.
  • Xi of HC CDR1 is N
  • X2 is N and X5 is W.
  • X4 of CDR1 when X4 of CDR1 is H, Xi is S and X5 is Y.
  • Xi of HC CDR1 when Xi of HC CDR1 is S, X2 is Y or G, and X3 is F or S. In some embodiments, when X3 of HC CDR1 is F, Xi is S and X5 is absent.
  • a CAR comprises a HC CDR2 having the amino acid sequence of X7IYX8X9GX10TNYNPSLKS (SEQ ID NO: 2), wherein X 7 is E, Y or F; X 8 is H, D, or Y; X9 is S or T; and X10 is S, N or T.
  • X7 of HC CDR2 is Y
  • Xs is Y or D
  • X10 is T or N.
  • Xs of HC CDR2 is Y
  • X7 is Y or F
  • X9 is S or T.
  • a CAR comprises a HC CDR3 having the amino acid sequence of X11X12X13X14IRGX15X16X17X18X19, wherein Xu is D or E; X12 is G, W, or absent; X13 is T, D, or E; X14 is M or L; X15 is V, A or H; Xi6 is G or absent; X17 is L or F; Xis is G or D; and X19 is Y, P, I, or D.
  • Xu of HC CDR3 is E
  • X12 is G or W
  • X15 is A or H
  • X19 is P, I, or D.
  • X12 of HC CDR3 when X12 of HC CDR3 is W, X13 is D or E, X15 is A or H, and X19 is I or D.
  • X14 of HC CDR3 when X14 of HC CDR3 is M, Xu is D or E, X12 is G or absent, X15 is V or A, and X19 is Y or P.
  • X14 when X14 is L, X13 is D or E, X15 is A or H, X19 is I or D.
  • a CAR comprises a LC CDR1 having the amino acid sequence of RASX20X21ISX22WLA (SEQ ID NO: 4), wherein X20 is Q or P, X21 is G or D, and X22 is S, R, or N.
  • a CAR comprises a LC CDR2 having the amino acid sequence of X23ASSLQX24 (SEQ ID NO: 5), wherein X23 is A, T or V, and X24 is S or G. In some embodiments, when X23 of LC CDR2 is A, X24 is S or G.
  • a CAR comprises a LC CDR3 having the amino acid sequence of QQX25NX26FPX27T (SEQ ID NO: 6), wherein X25 is T or A; X26 is N or S; and X27 is W or L.
  • SEQ ID NO: 6 QQX25NX26FPX27T
  • Xi of HC CDR1 is S
  • X2 is Y or G
  • X3 is F or S
  • X5 is T or absent
  • Xf> is S or N
  • X? is E, Y or F
  • Xs is H, D, or Y
  • X9 is S or T
  • X10 is S, N, or T
  • Xu is D or E
  • X12 is W or absent
  • X13 is T, D, or E
  • X14 is M or L
  • X15 is V, A or H
  • Xi6 is absent
  • X17 is L or F
  • Xis is G or D
  • X19 is Y, I, or D.
  • X23 is A or V
  • X24 is S or G
  • X25 is T or A
  • X27 is L.
  • Xi is N or S
  • X2 is N, Y, or G
  • X3 is Y, F, or S
  • X4 is H or absent
  • X5 is W, Y, or absent
  • Xi> is S
  • X7 is Y or F
  • Xs is Y or D
  • X9 is S or T
  • X10 is T or N
  • X12 is G or W
  • X13 is T, D, or E
  • X14 is M or L
  • X15 is A or H
  • Xi6 is G or absent
  • X17 is F
  • Xis is D
  • X19 is P, I, or D.
  • X23 is T, A, or V
  • X24 is S or G
  • X25 is A
  • X26 is S
  • X27 is L or W.
  • a CAR comprises a variable heavy domain (VH) with a framework region 1 (FR1) having the amino acid sequence of SEQ ID NO: 7, a framework region 2 (FR2) having the amino acid sequence of SEQ ID NO: 8, a framework region 3 (FR3) having the amino acid sequence of SEQ ID NO: 9, and a framework region 4 (FR4) having the amino acid sequence of SEQ ID NO: 10.
  • VH variable heavy domain
  • a CAR comprises a VH domain with a FR1 having the amino acid sequence of QVQLQESGPGLVKPSX28TLSLTCX29VSGX30SX31X32 (SEQ ID NO: 7), wherein X28 is G or E; X29 is T or A; X30 is G or D; X31 is I or V; and X32 is N or S.
  • a CAR comprises a VH domain with a FR2 having the amino acid sequence of WX33RQPPGKGLEWIG (SEQ ID NO: 8), wherein X33 is I or V.
  • a CAR comprises a VH domain with a FR3 having the amino acid sequence of RVTISX37DX35SKNX36FSLX37LX38SVTAADTAVYYCAR (SEQ ID NO: 9), wherein X37 is V or L, X35 is T, K, or P, X36 is Q or H, X37 is K or N, and X38 is S or R.
  • a CAR comprises a VH domain with a FR4 having the amino acid sequence of WGQGTX39VTVSS (SEQ ID NO: 10), wherein X39 is L or M.
  • the disclosure provides a chimeric antigen receptor (CAR) comprising a heavy chain complementarity determining region 1 (HC CDR1) comprising the amino acid sequence X40X41X40WX43, wherein X40 is S or T, X41 is Y or F, X40 is F or Y, and X43 is N or S; a heavy chain complementarity determining region 2 (HC CDR2) comprising the amino acid sequence YIYYSGX44TX45YNPSLKS (SEQ ID NO: 12), wherein X44 is S or T, and X45 is N or Y; and a heavy chain complementarity determining region 3 (HC CDR3) comprising the amino acid sequence X46X47X48LIRGX49X50
  • the CAR further comprises a light chain complementarity determining region 1 (LC CDR1) comprising the amino acid sequence RASQGISSWLA (SEQ ID NO: 24); a light chain complementarity determining region 2 (LC CDR2) comprising the amino acid sequence X52ASSLQS (SEQ ID NO: 14), wherein X52 is A or V; and a light chain complementarity determining region 3 (LC CDR3) comprising the amino acid sequence QQX53NX54FPLT (SEQ ID NO: 15), wherein X53 is T or A, and X54 is N or S.
  • LC CDR1 comprising the amino acid sequence RASQGISSWLA
  • LC CDR2 comprising the amino acid sequence X52ASSLQS (SEQ ID NO: 14), wherein X52 is A or V
  • LC CDR3 comprising the amino acid sequence QQX53NX54FPLT (SEQ ID NO: 15), wherein X53 is T or A, and X54 is N or S.
  • X44 is S
  • X40 is S
  • X41 is Y
  • X40 is F or Y
  • X43 is S
  • X45 is N or Y
  • X46 is D or E
  • X47 is W
  • X49 is L
  • X50 is H
  • X51 is L
  • X52 G or D
  • X53 is V
  • X54 is T or A
  • X55 is N or S.
  • X51 is F
  • X40 is S
  • X41 is Y
  • X40 is F or Y
  • X43 is S
  • X44 is T or S
  • X45 is Y or N
  • X46 is E
  • X47 is W
  • X48 is E
  • X50 is H
  • X52 is D
  • X53 is V
  • X54 is A
  • X55 is S.
  • X40 when X40 is T, X41 is F, X40 is F, X43 is N, X44 is T, X45 is N, X46 is D, X47 is T, X48 is M, X49 is absent, X50 is V, X51 is I, X52 is G, X53 is A, X54 is T, and X55 is N.
  • X40 when X40 is S, X41 is Y, X40 is F, X43 is N, X44 is T, X45 is N, X46 is D, X47 is T, X48 is M, X49 is absent, X50 is V, X51 is L, X52 is G, X53 is A, X54 is T, and X55 is N.
  • an anti-PRAME/HLA-A2 CAR comprises an extracellular ligand-binding domain that comprises a single-chain variable fragment (scFv).
  • the VH and the VL of an anti-PRAME/HLA-A2 CAR are joined together by a linker.
  • a linker may have a length of about 2 to 10 amino acids, 5 to 20 amino acids, 10 to 30 amino acids, 20-50 amino acids, 40 to 60 amino acids, 60 to 80 amino acids, or more than 80 amino acids.
  • a linker may include, without limitation, any of those encompassed by U.S. Patent Nos. 8,445,251 and 9,434,931.
  • an anti-PRAME/HLA-A2 CAR comprises an extracellular ligand-binding domain that comprises a scFv comprising a VH and a VL, and the C-terminus of the VH is joined with the N terminus of the VL via a linker.
  • an anti-PRAME/HLA-A2 CAR comprises an extracellular ligand-binding domain that comprises scFv comprising a VH and a VL, and the C-terminus of the VL is joined with the N terminus of the VH via a linker.
  • an anti-PRAME/HLA-A2 CAR of the present disclosure further comprises a hinge region.
  • hinge region can be used in the anti-PRAME/HLA-A2 CAR described herein, e.g., hinge regions derived from CD4, CD8, CD28, CD3, IgGl, IgG4, IgD, IgA, or IgM, a hybrid or variant therefore (see, e.g., Jayaraman et al., CAR-T design: Elements and their synergistic function, eBioMedicine, VOLUME 58, 102931, AUGUST 2020); Guedan et al., Engineering and Design of Chimeric Antigen Receptors, Mol Ther Methods Clin Dev. 2019 Mar 15; 12: 145-156).
  • an anti-PRAME/HLA-A2 CAR of the present disclosure further comprises a transmembrane domain, which links the extracellular ligand-binding domain with the intracellular signaling and co-stimulatory domains.
  • the CAR can be designed to comprise a transmembrane domain that is fused to the extracellular domain (e.g., the antigen binding domain) of the CAR directly or via the hinge region. Any transmembrane domain is contemplated for use herein as long as the domain is capable of anchoring a CAR comprising the domain to a cell membrane.
  • the transmembrane domain that naturally is associated with one of the domains in the CAR is used.
  • the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.
  • the transmembrane domain may be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. In some embodiments, the transmembrane domain may be synthetic, in which case it will comprise predominantly hydrophobic residues such as leucine and valine.
  • a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain.
  • a short oligo- or polypeptide linker preferably between 2 and 10 amino acids in length may form the linkage between the transmembrane domain and the cytoplasmic signaling domain of the CAR.
  • a glycine-serine doublet provides a particularly suitable linker.
  • the transmembrane domain can be any suitable transmembrane domain known in the art, e.g., transmembrane domain derived from TCRa, TCRp, TCR( ⁇ , CD3( ⁇ , CD3s, CD3y, CD35, CD4, CD5, CD8, CD9, CD16, CD22, CD28, CD32, CD33, CD34, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, or inducible T cell costimulator (ICOS).
  • any transmembrane domain is contemplated for use herein as long as the domain is capable of anchoring a CAR comprising the extracellular domain to a cell membrane.
  • Transmembrane domains can be identified using any method known in the art or described herein, e.g., by using the UniProt Database.
  • an anti-PRAME/HLA-A2 CAR further comprises an intracellular (or cytoplasmic) domain.
  • the intracellular domain of the CAR is responsible for activation of at least one of the normal effector functions of the immune cell in which the CAR has been placed in.
  • effector function refers to a specialized function of a cell (e.g., a T cell, a NK cell, or a NKT cell). Effector function of a cell (e.g., a T cell, a NK cell, or a NKT cell), for example, may be cytolytic activity or helper activity including the secretion of cytokines.
  • intracellular signaling domain refers to the portion of a protein which transduces the effector function signal and directs the cell to perform a specialized function. While usually the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire domain. To the extent that a truncated portion of the intracellular signaling domain is used, such truncated portion may be used in place of the intact domain as long as it transduces the effector function signal.
  • intracellular signaling domain is thus meant to include any truncated portion of the intracellular signaling domain sufficient to transduce the effector function signal.
  • the intracellular (or cytoplasmic) domain of a chimeric antigen receptor as disclosed herein may include, but is not limited to, a 4- IBB intracellular domain, a 0X40 intracellular domain, a CD30 intracellular domain, a CD40 intracellular domain, an ICOS intracellular domain, a LFA-1 intracellular domain, a CD2 intracellular domain, a CD3 C, intracellular domain, a CD3 y intracellular domain, a CD3 5 intracellular domain, a CD3 a intracellular domain, and a CD7 intracellular domain, and a CD22 intracellular domain.
  • the intracellular domain further comprises one or more intracellular co-stimulatory domains, such as those described herein, which transmit a costimulatory signal which promotes cell proliferation, cell survival, and/or cytokine secretion after binding of the extracellular domain.
  • intracellular co- stimulatory domains include, without limitation, any co-stimulatory domain disclosed herein or those domains known in the art, including but not limited to CD28, ICOS, 4- IBB, 0X40, or CD27.
  • the intracellular signaling domain of a chimeric antigen receptor of the present disclosure is responsible for activation of at least one of the normal effector functions of the cell in which the CAR has been placed and/or activation of proliferative and cell survival pathways.
  • a chimeric antigen receptor as disclosed herein can include a domain (e.g., an extracellular domain, a transmembrane domain, an intracellular (cytoplasmic) domain, a co-stimulatory domain, a signaling domain, or any combination thereof) having a sequence as set forth herein, or a variant thereof, or a fragment thereof, of any one or more of the domains disclosed herein (e.g., a variant and/or fragment that retains the function required for the chimeric antigen receptor activity).
  • the present disclosure provides genetically modified immune cells (e.g., T cells, NK cells, or NKT cells) expressing the anti-PRAME/HLA-A2 CAR described herein.
  • the present disclosure provides a multispecific (e.g., bispecific) antibody comprising one or more first antigen binding sites targeting PRAME/HLA-A2 complex, and one or more second binding sites targeting one or more different epitopes on a PRAME HLA-A2 complex.
  • a multispecific (e.g., bispecific antibody) comprising one or more first antigen binding sites targeting PRAME/HLA-A2 complex, and one or more second binding sites targeting one or more different antigens.
  • the present disclosure provides a bispecific antibody comprising a first antigen binding site targeting PRAME/HLA-A2 complex, and a second binding site.
  • the present disclosure provides a bispecific antibody comprising a first antigen binding site targeting PRAME/HLA-A2 complex, and a second antigen binding site specifically binds a different epitope on a PRAME HLA-A2 complex.
  • the present disclosure provides a bispecific antibody comprising a first antigen binding site targeting PRAME/HLA-A2 complex, and a second antigen binding site specifically binds a different epitope on a different antigen.
  • a bispecific antibody described herein comprises a first antigen binding site comprising an antigen binding site of any one of the anti-PRAME/HLA-A2 complex antibodies described in Table 1. In some embodiments, a bispecific antibody described herein comprises a first antigen binding site comprising a HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and/or LC CDR3 of any one of the anti-PRAME/HLA-A2 complex antibodies described in Table 1. In some embodiments, a bispecific antibody described herein comprises a first antigen binding site comprising a VH and/or VL of any one of the anti-PRAME/HLA-A2 complex antibodies described in Table 1.
  • a bispecific antibody described herein is a T cell engaging antibody that also targets PRAME/HLA-A2 complex.
  • a bispecific antibody described herein comprises a first antigen binding site that specifically binds a PRAME/HLA-A2 complex and a second antigen binding site that specifically binds a T cell antigen (referred to as an anti-PRAME/HLA-A2xT cell bispecific antibody.
  • an anti-PRAME/HLA-A2xT cell bispecific antibody comprises a second antigen binding site that specifically binds a T cell receptor specifically binds a CD3 complex.
  • the second antigen binding site specifically binds to CD3s of a CD3 complex.
  • the second antigen binding site specifically binds to CD3y of a CD3 complex. In some embodiments, the second antigen binding site specifically binds to CD35 of a CD3 complex. In some embodiments, the second antigen binding site specifically binds to a CD3s/5 heterodimer of a CD3 complex. In some embodiments, the second antigen binding site specifically binds to a CD3s/y heterodimer of a CD3 complex.
  • Any suitable antigen binding site that specifically binds to CD3 derived from an anti-CD3 antibody can be combined with an antigen binding site that specifically binds to a PRAME/HLA-A2 complex as described herein to form an anti-PRAME/HLA-A2xT cell bispecific antibody of the present disclosure.
  • an anti-PRAME/HLA- A2xT cell bispecific antibody comprises a first antigen binding site that specifically binds a PRAME/HLA-A2 complex (e.g., an antigen binding site derived from any one of the anti- PRAME/HLA-A2 complex described herein), and a second antigen binding site that specifically binds to CD3 that is derived from any one of the anti-CD3 antibodies previously described, e.g., in US10968276, US20060275292, WO1997044362, W02005118635, W02007042261, WO2011050262, WO2011078332, WO2012073985, WO2012158818, WO2012098238, WO2012162067, WO2013026835, WO2013026839, WO2013026833, WO2013026831, W02013065708, WO2014047231, W02014012085, WO2015095392, WO2015143079, W
  • a need for recruiting a T cell to a cancer cell expressing PRAME for killing the cancer cell exists.
  • the present disclosure provides means for engaging a T cell to a cancer cell expressing PRAME.
  • the means for engaging a T cell to a cancer cell expressing PRAME is an anti- PRAME/HLA-A2xT cell bispecific antibody described herein.
  • the present disclosure provides means for eliciting cytotoxic T cell immunity against cancer cells expressing PRAME.
  • the means for eliciting cytotoxic T cell immunity against cancer cells expressing PRAME is an anti-PRAME/HLA-A2xT cell bispecific antibody described herein.
  • an anti-PRAME/HLA-A2xT cell bispecific antibody comprises a second antigen binding site that specifically binds to CD3, wherein the improvement comprises a first antigen binding site that specifically binds to a PRAME/HLA- A2 complex that comprises any one of the anti-PRAME/A2 binding site derived from any of the anti-PRAME/A2 antibodies described herein (e.g., any one of the anti-PRAME/HLA-A2 antibody described in Table 1).
  • an anti-PRAME/HLA-A2xT cell bispecific antibody described herein comprises a first antigen binding site that specifically binds to a PRAME/HLA-A2 complex.
  • an anti-PRAME/HLA-A2xT cell bispecific antibody described herein comprises a first antigen binding site comprising a HC CDR1, a HC CDR2 and a HC CDR3 of a heavy chain variable domain (VH) having the amino acid sequence of antibody #1 in row 34; antibody #2 in row 35, antibody #3 in row 36, antibody #4 in row 37, antibody #5 in row 38, antibody #6 in row 39, antibody #7 in row 40, antibody #8 in row 41, antibody #9 in row 42, antibody #10 in row 43, antibody #11 in row 44, antibody #12 in row 45, antibody #13 in row 46, antibody #14 in row 47, antibody #15 in row 48, antibody #16 in row 49, antibody #17 in row 50, antibody #18 in row 51, antibody #19 in
  • an anti-PRAME/HLA-A2xT cell bispecific antibody described herein comprises a first antigen binding site comprising a LC CDR1, a LC CDR2 and a LC CDR3 of a light chain variable domain (VL) having the amino acid sequence of antibody #1 in row 34; antibody #2 in row 35, antibody #3 in row 36, antibody #4 in row 37, antibody #5 in row 38, antibody #6 in row 39, antibody #7 in row 40, antibody #8 in row 41, antibody #9 in row 42, antibody #10 in row 43, antibody #11 in row 44, antibody #12 in row 45, antibody #13 in row 46, antibody #14 in row 47, antibody #15 in row 48, antibody #16 in row 49, antibody #17 in row 50, antibody #18 in row 51, antibody #19 in row 52, antibody #20 in row 53, antibody #21 in row 54, antibody #22 in row 55, antibody #23 in row 56, antibody #24 in row 57, antibody #25 in for 58, antibody #26 in row 59
  • VL light
  • an anti-PRAME/HLA-A2xT cell bispecific antibody described herein comprises a first antigen binding site comprising a HC CDR3 having the amino acid sequence of antibody #1 in row 1, antibody #2 in row 2, antibody #3 in row 3, antibody #4 in row 4, antibody #5 in row 5, antibody #6 in row 6, antibody #7 in row 7, antibody #8 in row 8, antibody #9 in row 9, antibody #10 in row 10, antibody #11 in row 11, antibody #12 in row 12, antibody #13 in row 13, antibody #14 in row 14, antibody, antibody #15 in row 15, antibody #16 in row 16, antibody #17 in row 17, antibody #18 in row 18, antibody #19 in row 19, antibody #20 in row 20, antibody #21 in row 21, antibody #22 in row 22, antibody #23 in row 23, antibody #24 in row 24, antibody #25 in row 25, antibody #26 in row 26, antibody #27 in row 27 antibody #28 in row 28, antibody #29 in row 29, antibody #30 in row 30, antibody #31 in row 31, antibody #32 in row 32, or antibody #33 in row 33, column A of
  • an anti-PRAME/HLA-A2xT cell bispecific antibody described herein comprises a first antigen binding site comprising a HC CDR1, a HC CDR2, and a HC CDR3, having the amino acid sequences of antibody #1 in row 1, antibody #2 in row 2, antibody #3 in row 3, antibody #4 in row 4, antibody #5 in row 5, antibody #6 in row 6, antibody #7 in row 7, antibody #8 in row 8, antibody #9 in row 9, antibody #10 in row 10, antibody #11 in row 11, antibody #12 in row 12, antibody #13 in row 13, antibody #14 in row 14, antibody, antibody #15 in row 15, antibody #16 in row 16, antibody #17 in row 17, antibody #18 in row 18, antibody #19 in row 19, antibody #20 in row 20, antibody #21 in row 21, antibody #22 in row 22, antibody #23 in row 23, antibody #24 in row 24, antibody #25 in row 25, antibody #26 in row 26, antibody #27 in row 27 antibody #28 in row 28, antibody #29 in row 29, antibody #30 in row 30, antibody #31 in row 31, antibody #32 in row 32,
  • an anti-PRAME/HLA-A2xT cell bispecific antibody described herein comprises a first antigen binding site comprising a LC CDR, a LC CDR2, and a LC CDR3 having the amino acid sequence of antibody #1 in row 1, antibody #2 in row 2, antibody #3 in row 3, antibody #4 in row 4, antibody #5 in row 5, antibody #6 in row 6, antibody #7 in row 7, antibody #8 in row 8, antibody #9 in row 9, antibody #10 in row 10, antibody #11 in row 11, antibody #12 in row 12, antibody #13 in row 13, antibody #14 in row 14, antibody, antibody #15 in row 15, antibody #16 in row 16, antibody #17 in row 17, antibody #18 in row 18, antibody #19 in row 19, antibody #20 in row 20, antibody #21 in row 21, antibody #22 in row 22, antibody #23 in row 23, antibody #24 in row 24, antibody #25 in row 25, antibody #26 in row 26, antibody #27 in row 27 antibody #28 in row 28, antibody #29 in row 29, antibody #30 in row 30, antibody #31 in row 31, antibody #31 in row 31, antibody #31 in row 31,
  • an anti-PRAME/HLA-A2xT cell bispecific antibody described herein comprises a first antigen binding site comprising a HC CDR1, a HC CDR2, a HC CDR3, a LC CDR1, a LC CDR2, and a LC CDR3 having the amino acid sequence of antibody #1 in row 1, antibody #2 in row 2, antibody #3 in row 3, antibody #4 in row 4, antibody #5 in row 5, antibody #6 in row 6, antibody #7 in row 7, antibody #8 in row 8, antibody #9 in row 9, antibody #10 in row 10, antibody #11 in row 11, antibody #12 in row 12, antibody #13 in row 13, antibody #14 in row 14, antibody, antibody #15 in row 15, antibody #16 in row 16, antibody #17 in row 17, antibody #18 in row 18, antibody #19 in row 19, antibody #20 in row 20, antibody #21 in row 21, antibody #22 in row 22, antibody #23 in row 23, antibody #24 in row 24, antibody #25 in row 25, antibody #26 in row 26, antibody #27 in row 27 antibody #28 in row 28, antibody #29 in row
  • an anti-PRAME/HLA-A2xT cell bispecific antibody described herein comprises a first antigen binding site comprising comprises a HC CDR1, a HC CDR2, and a HC CDR3, which collectively contains no more than 5 amino acid variations (e.g., no more than 5, 4, 3, 2, or 1 amino acid variation) as compared with the HC CDR1, the HC CDR2, and the HC CDR3 amino acid sequences of antibody #1 in row 1, antibody #2 in row 2, antibody #3 in row 3, antibody #4 in row 4, antibody #5 in row 5, antibody #6 in row 6, antibody #7 in row 7, antibody #8 in row 8, antibody #9 in row 9, antibody #10 in row 10, antibody #11 in row 11, antibody #12 in row 12, antibody #13 in row 13, antibody #14 in row 14, antibody, antibody #15 in row 15, antibody #16 in row 16, antibody #17 in row 17, antibody #18 in row 18, antibody #19 in row 19, antibody #20 in row 20, antibody #21 in row 21, antibody #22 in row 22, antibody #23 in row
  • an anti-PRAME/HLA-A2xT cell bispecific antibody described herein comprises a first antigen binding site comprising a LC CDR1, a LC CDR2, and a LC CDR3, which collectively contains no more than 5 amino acid variations (e.g., no more than 5, 4, 3, 2 or 1 amino acid variation) as compared with the LC CDR1, the LC CDR2, and the LC CDR3 amino acid sequences of antibody #1 in row 1, antibody #2 in row 2, antibody #3 in row 3, antibody #4 in row 4, antibody #5 in row 5, antibody #6 in row 6, antibody #7 in row 7, antibody #8 in row 8, antibody #9 in row 9, antibody #10 in row 10, antibody #11 in row 11, antibody #12 in row 12, antibody #13 in row 13, antibody #14 in row 14, antibody, antibody #15 in row 15, antibody #16 in row 16, antibody #17 in row 17, antibody #18 in row 18, antibody #19 in row 19, antibody #20 in row 20, antibody #21 in row 21, antibody #22 in row 22, antibody #23 in row 23, antibody #24
  • an anti-PRAME/HLA-A2xT cell bispecific antibody described herein comprises a first antigen binding site comprising a HC CDR1, a HC CDR2, and a HC CDR3 that collectively are at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the HC CDR1, the HC CDR2, and the HC CDR3 amino acid sequences of antibody #1 in row 1, antibody #2 in row 2, antibody #3 in row 3, antibody #4 in row 4, antibody #5 in row 5, antibody #6 in row 6, antibody #7 in row 7, antibody #8 in row 8, antibody #9 in row 9, antibody #10 in row 10, antibody #11 in row 11, antibody #12 in row 12, antibody #13 in row 13, antibody #14 in row 14, antibody, antibody #15 in row 15, antibody #16
  • an anti-PRAME/HLA-A2xT cell bispecific antibody described herein comprises a first antigen binding site comprising a LC CDR1, a LC CDR2, and a LC CDR3 that collectively are at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the to the LC CDR1, the LC CDR2, and the LC CDR3 amino acid sequences of antibody #1 in row 1, antibody #2 in row 2, antibody #3 in row 3, antibody #4 in row 4, antibody #5 in row 5, antibody #6 in row 6, antibody #7 in row 7, antibody #8 in row 8, antibody #9 in row 9, antibody #10 in row 10, antibody #11 in row 11, antibody #12 in row 12, antibody #13 in row 13, antibody #14 in row 14, antibody, antibody #15 in row 15, antibody #16 in row
  • an anti-PRAME/HLA-A2xT cell bispecific antibody described herein comprises a first antigen binding site comprising a HC CDR1 having no more than 3 amino acid variations (e.g., no more than 3, 2, or 1 amino acid variation); a HC CDR2 having no more than 3 amino acid variations (e.g., no more than 3, 2, or 1 amino acid variation); and/or a HC CDR3 having no more than 3 amino acid variations (e.g., no more than 3, 2, or 1 amino acid variation) as compared with the HCCDR1, the HC CDR2, and the HC CDR3 amino acid sequences of antibody #1 in row 1, antibody #2 in row 2, antibody #3 in row 3, antibody #4 in row 4, antibody #5 in row 5, antibody #6 in row 6, antibody #7 in row 7, antibody #8 in row 8, antibody #9 in row 9, antibody #10 in row 10, antibody #11 in row 11, antibody #12 in row 12, antibody #13 in row 13, antibody #14 in row 14, antibody, antibody #15 in row 15, antibody
  • an anti-PRAME/HLA-A2xT cell bispecific antibody described herein comprises a first antigen binding site comprising a LC CDR1 having no more than 3 amino acid variations (e.g., no more than 3, 2, or 1 amino acid variation); a LC CDR2 having no more than 3 amino acid variations (e.g., no more than 3, 2, or 1 amino acid variation); and/or a LC CDR3 having no more than 3 amino acid variations (e.g., no more than 3, 2, or 1 amino acid variation) as compared with the LC CDR1, the LC CDR2, and the LC CDR3 amino acid sequences of antibody #1 in row 1, antibody #2 in row 2, antibody #3 in row 3, antibody #4 in row 4, antibody #5 in row 5, antibody #6 in row 6, antibody #7 in row 7, antibody #8 in row 8, antibody #9 in row 9, antibody #10 in row 10, antibody #11 in row 11, antibody #12 in row 12, antibody #13 in row 13, antibody #14 in row 14, antibody, antibody #15 in row 15, antibody #16 in
  • an anti-PRAME/HLA-A2xT cell bispecific antibody described herein comprises a first antigen binding site comprising a HC CDR1 at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the HC CDR1; a HC CDR2 at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the HC CDR2; and/or a HC CDR3 at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%,
  • an anti- PRAME/HLA-A2xT cell bispecific antibody described herein comprises a first antigen binding site comprising a LC CDR1 at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the LC CDR1; a LC CDR2 at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the LC CDR2,; and/or a LC CDR3 at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%
  • an anti-PRAME/HLA-A2xT cell bispecific antibody described herein comprises a first antigen binding site comprising a VH comprising the amino acid sequence of antibody #1 in row 34; antibody #2 in row 35, antibody #3 in row 36, antibody #4 in row 37, antibody #5 in row 38, antibody #6 in row 39, antibody #7 in row 40, antibody #8 in row 41, antibody #9 in row 42, antibody #10 in row 43, antibody #11 in row 44, antibody #12 in row 45, antibody #13 in row 46, antibody #14 in row 47, antibody #15 in row 48, antibody #16 in row 49, antibody #17 in row 50, antibody #18 in row 51, antibody #19 in row 52, antibody #20 in row 53, antibody #21 in row 54, antibody #22 in row 55, antibody #23 in row 56, antibody #24 in row 57, antibody #25 in for 58, antibody #26 in row 59, antibody #27 in row 60, antibody #28 in row 61, antibody #29 in row 62,
  • an anti-PRAME/HLA-A2xT cell bispecific antibody described herein comprises a first antigen binding site comprising a VL comprising the amino acid sequence of antibody #1 in row 34; antibody #2 in row 35, antibody #3 in row 36, antibody #4 in row 37, antibody #5 in row 38, antibody #6 in row 39, antibody #7 in row 40, antibody #8 in row 41, antibody #9 in row 42, antibody #10 in row 43, antibody #11 in row 44, antibody #12 in row 45, antibody #13 in row 46, antibody #14 in row 47, antibody #15 in row 48, antibody #16 in row 49, antibody #17 in row 50, antibody #18 in row 51, antibody #19 in row 52, antibody #20 in row 53, antibody #21 in row 54, antibody #22 in row 55, antibody #23 in row 56, antibody #24 in row 57, antibody #25 in for 58, antibody #26 in row 59, antibody #27 in row 60, antibody #28 in row 61, antibody #29 in row 62, antibody #30 in row
  • an anti-PRAME/HLA-A2xT cell bispecific antibody described herein comprises a first antigen binding site comprising a VH and a VL comprising the amino acid sequences of antibody #1 in row 34; antibody #2 in row 35, antibody #3 in row 36, antibody #4 in row 37, antibody #5 in row 38, antibody #6 in row 39, antibody #7 in row 40, antibody #8 in row 41, antibody #9 in row 42, antibody #10 in row 43, antibody #11 in row 44, antibody #12 in row 45, antibody #13 in row 46, antibody #14 in row 47, antibody #15 in row 48, antibody #16 in row 49, antibody #17 in row 50, antibody #18 in row 51, antibody #19 in row 52, antibody #20 in row 53, antibody #21 in row 54, antibody #22 in row 55, antibody #23 in row 56, antibody #24 in row 57, antibody #25 in for 58, antibody #26 in row 59, antibody #27 in row 60, antibody #28 in row 61, antibody #29 in row 62
  • an anti-PRAME/HLA-A2xT cell bispecific antibody described herein comprises a first antigen binding site comprising a VH containing no more than 20 amino acid variations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) as compared with the VH as set forth in antibody #1 in row 34; antibody #2 in row 35, antibody #3 in row 36, antibody #4 in row 37, antibody #5 in row 38, antibody #6 in row 39, antibody #7 in row 40, antibody #8 in row 41, antibody #9 in row 42, antibody #10 in row 43, antibody #11 in row 44, antibody #12 in row 45, antibody #13 in row 46, antibody #14 in row 47, antibody #15 in row 48, antibody #16 in row 49, antibody #17 in row 50, antibody #18 in row 51, antibody #19 in row 52, antibody #20 in row 53, antibody #21 in row 54, antibody #22 in row 55, antibody #23 in row 56,
  • VH containing no more than
  • an anti-PRAME/HLA-A2xT cell bispecific antibody described herein comprises a first antigen binding site comprising a VL containing no more than 20 amino acid variations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) as compared with the VL as set forth in antibody #1 in row 34; antibody #2 in row 35, antibody #3 in row 36, antibody #4 in row 37, antibody #5 in row 38, antibody #6 in row 39, antibody #7 in row 40, antibody #8 in row 41, antibody #9 in row 42, antibody #10 in row 43, antibody #11 in row 44, antibody #12 in row 45, antibody #13 in row 46, antibody #14 in row 47, antibody #15 in row 48, antibody #16 in row 49, antibody #17 in row 50, antibody #18 in row 51, antibody #19 in row 52, antibody #20 in row 53, antibody #21 in row 54, antibody #22 in row 55, antibody #23 in row 56, antibody #24 in row
  • VL
  • an anti-PRAME/HLA-A2xT cell bispecific antibody described herein comprises a first antigen binding site comprising a VH comprising an amino acid sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VH as set forth in the amino acid sequence of antibody #1 in row 34; antibody #2 in row 35, antibody #3 in row 36, antibody #4 in row 37, antibody #5 in row 38, antibody #6 in row 39, antibody #7 in row 40, antibody #8 in row 41, antibody #9 in row 42, antibody #10 in row 43, antibody #11 in row 44, antibody #12 in row 45, antibody #13 in row 46, antibody #14 in row 47, antibody #15 in row 48, antibody #16 in row 49, antibody #17 in row 50, antibody #18
  • an anti-PRAME/HLA-A2xT cell bispecific antibody described herein comprises a first antigen binding site comprising a VL comprising an amino acid sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VL as set forth in the amino acid sequence of antibody #1 in row 34; antibody #2 in row 35, antibody #3 in row 36, antibody #4 in row 37, antibody #5 in row 38, antibody #6 in row 39, antibody #7 in row 40, antibody #8 in row 41, antibody #9 in row 42, antibody #10 in row 43, antibody #11 in row 44, antibody #12 in row 45, antibody #13 in row 46, antibody #14 in row 47, antibody #15 in row 48, antibody #16 in row 49, antibody #17 in row 50, antibody #18 in row 51
  • an anti-PRAME/HLA-A2xT cell bispecific antibody described herein comprises a first antigen binding site comprising a comprises a VH containing no more than 20 amino acid variations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) as compared with the VH as set forth in the amino acid sequence of antibody #1 in row 34; antibody #2 in row 35, antibody #3 in row 36, antibody #4 in row 37, antibody #5 in row 38, antibody #6 in row 39, antibody #7 in row 40, antibody #8 in row 41, antibody #9 in row 42, antibody #10 in row 43, antibody #11 in row 44, antibody #12 in row 45, antibody #13 in row 46, antibody #14 in row 47, antibody #15 in row 48, antibody #16 in row 49, antibody #17 in row 50, antibody #18 in row 51, antibody #19 in row 52, antibody #20 in row 53, antibody #21 in row 54, antibody #22 in row 55
  • an anti-PRAME/HLA-A2xT cell bispecific antibody described herein comprises a first antigen binding site comprising a VL containing no more than 20 amino acid variations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) as compared with the VL as set forth in the amino acid sequence of antibody #1 in row 34; antibody #2 in row 35, antibody #3 in row 36, antibody #4 in row 37, antibody #5 in row 38, antibody #6 in row 39, antibody #7 in row 40, antibody #8 in row 41, antibody #9 in row 42, antibody #10 in row 43, antibody #11 in row 44, antibody #12 in row 45, antibody #13 in row 46, antibody #14 in row 47, antibody #15 in row 48, antibody #16 in row 49, antibody #17 in row 50, antibody #18 in row 51, antibody #19 in row 52, antibody #20 in row 53, antibody #21 in row 54, antibody #22 in row 55, antibody #23 in row 56,
  • VL
  • the number of amino acid variations may occur within a VH of and/or a VL of the amino acid sequences of antibody #1 in row 34; antibody #2 in row 35, antibody #3 in row 36, antibody #4 in row 37, antibody #5 in row 38, antibody #6 in row 39, antibody #7 in row 40, antibody #8 in row 41, antibody #9 in row 42, antibody #10 in row 43, antibody #11 in row 44, antibody #12 in row 45, antibody #13 in row 46, antibody #14 in row 47, antibody #15 in row 48, antibody #16 in row 49, antibody #17 in row 50, antibody #18 in row 51, antibody #19 in row 52, antibody #20 in row 53, antibody #21 in row 54, antibody #22 in row 55, antibody #23 in row 56, antibody #24 in row 57, antibody #25 in for 58, antibody #26 in row 59, antibody #27 in row 60,
  • an anti- PRAME/HLA-A2xT cell bispecific antibody described herein comprises a first antigen binding site comprising a heavy chain variable sequence that comprises a framework sequence that that contains no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation to the framework sequence of a VH of antibody #1 in row 34; antibody #2 in row 35, antibody #3 in row 36, antibody #4 in row 37, antibody #5 in row 38, antibody #6 in row 39, antibody #7 in row 40, antibody #8 in row 41, antibody #9 in row 42, antibody #10 in row 43, antibody #11 in row 44, antibody #12 in row 45, antibody #13 in row 46, antibody #14 in row 47, antibody #15 in row 48, antibody #16 in row 49, antibody #17 in row 50, antibody #18 in row 51, antibody #19 in row 52, antibody #20 in row 53, antibody #21 in row 54, antibody #22 in row 55, antibody #23 in row 56, antibody #24 in row 57, antibody #25
  • an anti-PRAME/HLA-A2xT cell bispecific antibody described herein comprises a first antigen binding site comprising a VH comprising an amino acid sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VH as set forth in antibody #1 in row 34; antibody #2 in row 35, antibody #3 in row 36, antibody #4 in row 37, antibody #5 in row 38, antibody #6 in row 39, antibody #7 in row 40, antibody #8 in row 41, antibody #9 in row 42, antibody #10 in row 43, antibody #11 in row 44, antibody #12 in row 45, antibody #13 in row 46, antibody #14 in row 47, antibody #15 in row 48, antibody #16 in row 49, antibody #17 in row 50, antibody #18 in row 51, antibody
  • an anti- PRAME/HLA-A2xT cell bispecific antibody described herein comprises a first antigen binding site comprising a VL comprising an amino acid sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VL as set forth in antibody #1 in row 34; antibody #2 in row 35, antibody #3 in row 36, antibody #4 in row 37, antibody #5 in row 38, antibody #6 in row 39, antibody #7 in row 40, antibody #8 in row 41, antibody #9 in row 42, antibody #10 in row 43, antibody #11 in row 44, antibody #12 in row 45, antibody #13 in row 46, antibody #14 in row 47, antibody #15 in row
  • antibody #16 in row 49 antibody #17 in row 50, antibody #18 in row 51, antibody #19 in row 52, antibody #20 in row 53, antibody #21 in row 54, antibody #22 in row 55, antibody #23 in row 56, antibody #24 in row 57, antibody #25 in for 58, antibody #26 in row 59, antibody #27 in row 60, antibody #28 in row 61, antibody #29 in row 62, antibody #30 in row
  • the degree of sequence variation (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) may occur within a VH of antibody #1 in row 34; antibody #2 in row 35, antibody #3 in row 36, antibody #4 in row 37, antibody #5 in row 38, antibody #6 in row 39, antibody #7 in row 40, antibody #8 in row 41, antibody #9 in row 42, antibody #10 in row 43, antibody #11 in row 44, antibody #12 in row 45, antibody #13 in row 46, antibody #14 in row 47, antibody #15 in row 48, antibody #16 in row
  • antibody #17 in row 50 antibody #18 in row 51, antibody #19 in row 52, antibody #20 in row 53, antibody #21 in row 54, antibody #22 in row 55, antibody #23 in row 56, antibody #24 in row 57, antibody #25 in for 58, antibody #26 in row 59, antibody #27 in row 60, antibody #28 in row 61, antibody #29 in row 62, antibody #30 in row 63, antibody #31 in row
  • an anti-PRAME/HLA-A2xT cell bispecific antibody described herein comprises a first antigen binding site comprising a heavy chain variable sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the framework sequence of a VH of antibody #1 in row 34; antibody #2 in row 35, antibody #3 in row 36, antibody #4 in row 37, antibody #5 in row 38, antibody #6 in row 39, antibody #7 in row 40, antibody #8 in row 41, antibody #9 in row 42, antibody #10 in row 43, antibody #11 in row 44, antibody #12 in row 45, antibody #13 in row 46, antibody #14 in row 47, antibody #15 in row 48, antibody #16 in row 49, antibody #17 in row 50, antibody #18 in row 51, antibody #19 in row 52,
  • an anti-PRAME/HLA-A2xT cell bispecific antibody described herein comprises a second antigen binding site that specifically binds to CD3.
  • each of the two antigen binding sites is present within an “arm” of a bispecific antibody.
  • an arm of a bispecific antibody is configured as a monospecific antibody, e.g., a full-length IgG or a fragment thereof.
  • a bispecific antibody comprises two arms, one or each of which is configured as an Fv region that confers specificity to distinct antigen residues.
  • a bispecific antibody comprises two arms of the same configuration in any other suitable format.
  • a bispecific antibody comprises two arms of two different configurations.
  • an anti-PRAME/HEA-A2xT cell bispecific antibody comprises a first arm that is configured as a Fab, a Fab’, or a scFv and that comprises the first antigen binding site.
  • an anti-PRAME/HEA-A2xT cell bispecific antibody comprises a second arm that is configured as a Fab, a Fab’, or a scFv and that comprises the second antigen binding site.
  • an anti-PRAME/HEA-A2xT cell bispecific antibody comprises a first arm that is configured as an scFv that comprises a first antigen binding site (e.g., a first antigen binding site that specifically binds PRAME/HEA-A2 complex).
  • an anti-PRAME/HEA-A2xT cell bispecific antibody comprises a first arm that is configured as a Fab that comprises a first antigen binding site (e.g., a first antigen binding site that specifically binds PRAME/HEA-A2 complex).
  • an anti-PRAME/HEA-A2xT cell bispecific antibody comprises a first arm that is configured as a Fab’ that comprises a first antigen binding site (e.g., a first antigen binding site that specifically binds PRAME/HEA-A2 complex).
  • an anti- PRAME/HEA-A2xT cell bispecific antibody comprises a second arm that is configured as an scFv that comprises a second antigen binding site (e.g., a second antigen binding site that specifically binds CD3).
  • an anti-PRAME/HEA-A2xT cell bispecific antibody comprises a second arm that is configured as a Fab that comprises a second antigen binding site (e.g., a second antigen binding site that specifically binds CD3).
  • an anti-PRAME/HEA-A2xT cell bispecific antibody comprises a second arm that is configured as a Fab’ that comprises a second antigen binding site (e.g., a second antigen binding site that specifically binds CD3).
  • an anti-PRAME/HEA-A2xT cell bispecific antibody comprises a first arm that is configured as an scFv that comprises a first antigen binding site (e.g., a first antigen binding site that specifically binds PRAME/HEA-A2 complex) and a second arm that is configured as an scFv that comprises a second antigen binding site (e.g., a second antigen binding site that specifically binds CD3).
  • a first antigen binding site e.g., a first antigen binding site that specifically binds PRAME/HEA-A2 complex
  • a second arm that is configured as an scFv that comprises a second antigen binding site (e.g., a second antigen binding site that specifically binds CD3).
  • an anti- PRAME/HEA-A2xT cell bispecific antibody comprises a first arm that is configured as an scFv that comprises a first antigen binding site (e.g., a first antigen binding site that specifically binds PRAME/HEA-A2 complex), and a second arm that is configured as a Fab that comprises a second antigen binding site (e.g., a second antigen binding site that specifically binds CD3).
  • a first antigen binding site e.g., a first antigen binding site that specifically binds PRAME/HEA-A2 complex
  • a second arm that is configured as a Fab that comprises a second antigen binding site (e.g., a second antigen binding site that specifically binds CD3).
  • an anti-PRAME/HEA-A2xT cell bispecific antibody comprises a first arm that is configured as an scFv that comprises a first antigen binding site (e.g., a first antigen binding site that specifically binds PRAME/HEA-A2 complex), and a second arm that is configured as a Fab’ that comprises a second antigen binding site (e.g., a second antigen binding site that specifically binds CD3).
  • an anti-PRAME/HEA-A2xT cell bispecific antibody comprises a first arm that is configured as a Fab that comprises a first antigen binding site (e.g., a first antigen binding site that specifically binds PRAME/HLA-A2 complex), and a second arm that is configured as a Fab’ that comprises a second antigen binding site (e.g., a second antigen binding site that specifically binds CD3).
  • a first antigen binding site e.g., a first antigen binding site that specifically binds PRAME/HLA-A2 complex
  • a second arm that is configured as a Fab’ that comprises a second antigen binding site (e.g., a second antigen binding site that specifically binds CD3).
  • an anti-PRAME/HLA-A2xT cell bispecific antibody comprises a first arm that is configured as a Fab that comprises a first antigen binding site (e.g., a first antigen binding site that specifically binds PRAME/HEA-A2 complex), and a second arm that is configured as an scFv that comprises a second antigen binding site (e.g., a second antigen binding site that specifically binds CD3).
  • an anti-PRAME/HEA-A2xT cell bispecific antibody comprises a first arm that is configured as a Fab that comprises a first antigen binding site (e.g., a first antigen binding site that specifically binds PRAME/HEA-A2 complex), and a second arm that is configured as a Fab that comprises a second antigen binding site (e.g., a second antigen binding site that specifically binds CD3).
  • a first antigen binding site e.g., a first antigen binding site that specifically binds PRAME/HEA-A2 complex
  • a second arm that is configured as a Fab that comprises a second antigen binding site (e.g., a second antigen binding site that specifically binds CD3).
  • an anti-PRAME/HEA-A2xT cell bispecific antibody comprises a first arm that is configured as a Fab’ that comprises a first antigen binding site (e.g., a first antigen binding site that specifically binds PRAME/HEA-A2 complex), and a second arm that is configured as a Fab’ that comprises a second antigen binding site (e.g., a second antigen binding site that specifically binds CD3) that is a Fab’.
  • a first antigen binding site e.g., a first antigen binding site that specifically binds PRAME/HEA-A2 complex
  • a second arm that is configured as a Fab’ that comprises a second antigen binding site (e.g., a second antigen binding site that specifically binds CD3) that is a Fab’.
  • an anti-PRAME/HEA-A2xT cell bispecific antibody comprises a first arm that is configured as a Fab’ that comprises a first antigen binding site (e.g., a first antigen binding site that specifically binds PRAME/HEA-A2 complex), and a second arm that is configured as a Fab that comprises a second antigen binding site (e.g., a second antigen binding site that specifically binds CD3).
  • a Fab that comprises a first antigen binding site (e.g., a first antigen binding site that specifically binds PRAME/HEA-A2 complex)
  • a second arm that is configured as a Fab that comprises a second antigen binding site (e.g., a second antigen binding site that specifically binds CD3).
  • an anti-PRAME/HEA-A2xT cell bispecific antibody comprises a first arm that is configured as a Fab’ that comprises a first antigen binding site (e.g., a first antigen binding site that specifically binds PRAME/HEA-A2 complex), and a second arm that is configured as an scFv that comprises a second antigen binding site (e.g., a second antigen binding site that specifically binds CD3).
  • an anti-PRAME/HEA-A2xT cell bispecific antibody does not comprise an Fc region.
  • the two arms of an anti-PRAME/HEA- A2xT cell bispecific antibody are linked directly.
  • an anti-PRAME/HEA-A2xT cell bispecific antibody comprises an Fc region, e.g., a dimeric Fc or a monomeric Fc.
  • an anti-PRAME/HEA-A2xT cell bispecific antibody comprises a monomeric Fc.
  • an anti- PRAME/HLA-A2xT cell bispecific antibody comprises one arm linked to one end (e.g., the N terminal) of a monomeric Fc and a second arm linked to the other end (e.g., the C terminal) of the monomeric Fc.
  • an anti-PRAME/HLA-A2xT cell bispecific antibody comprises a dimeric Fc region. In some embodiments, an anti-PRAME/HLA-A2xT cell bispecific antibody comprises two arms that are oriented symmetrically around an Fc region (e.g., each Fc monomer of a dimeric Fc region is linked to one arm). In some embodiments, an anti-PRAME/HLA-A2xT cell bispecific antibody comprises two distinct heavy chains and two distinct light chains, with each heavy chain/light chain pair having different antigen binding specificity.
  • an anti-PRAME/HLA-A2xT cell bispecific antibody comprises two arms that are oriented asymmetrically around an Fc (e.g., the two arms of the anti-PRAME/HLA-A2xT cell bispecific antibody are linked to one of the monomers of a dimeric Fc region).
  • an anti-PRAME/HLA-A2x T cell bispecific antibody comprises a monomeric Fc containing mutations which abolish Fc gamma receptor (FcyR) binding, resulting in loss of effector functions in immune cells that would typically bind FcyRs, such as phagocytosis and cytokine release.
  • an anti- PRAME/HLA-A2xT cell bispecific antibody comprises a monomeric human serum albumin (HSA).
  • HSA human serum albumin
  • an anti-PRAME/HLA-A2x T cell bispecific antibody comprises an anti-HSA domain.
  • an anti-PRAME/HLA-A2x T cell bispecific antibody comprises an anti-HSA domain configured as a VHH antibody.
  • the ratio of the first antigen binding site (e.g., a first antigen binding site that specifically binds PRAME/HLA-A2 complex) and a second antigen binding site (e.g., a second antigen binding site that specifically binds CD3) of an anti-PRAME/HLA- A2xT cell bispecific antibody is 1:1, 1:2, 1:3, 1:4, 1:5, 2:3, 2:5, 3:4, 3:5, 4:5, 5:4, 5:3, 5:2, 3:2, 5:1, 4:1, 3:1, or 2:1.
  • an anti-PRAME/HLA-A2xT cell bispecific antibody comprises a first Fc region and a second Fc region.
  • an anti- PRAME/HLA-A2xT cell bispecific antibody does not include an Fc region.
  • an anti-PRAME/HLA-A2xT cell bispecific antibody can be constructed and assembled using any suitable methods into any suitable configurations, e.g., methods and configurations described by Ma et al., Bispecific Antibodies: From Research to Clinical Application, Front. Immunol., 05 May 2021, Sec. Cancer Immunity and Immunotherapy, Volume 12 - 2021, the entire contents are incorporated herein by reference. III. Preparation of the Anti-PRAME/HLA-A2 Antibodies
  • Antibodies capable of binding PRAME/HLA-A2 as described herein can be made by any method known in the art. See, for example, Harlow and Lane, (1998) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York.
  • antibodies specific to a target antigen can be made by the conventional hybridoma technology.
  • the full-length target antigen or a fragment thereof, optionally coupled to a carrier protein such as KLH, can be used to immunize a host animal for generating antibodies binding to that antigen.
  • the route and schedule of immunization of the host animal are generally in keeping with established and conventional techniques for antibody stimulation and production, as further described herein. General techniques for production of mouse, humanized, and human antibodies are known in the art and are described herein.
  • any mammalian subject including humans or antibody producing cells therefrom can be manipulated to serve as the basis for production of mammalian, including human hybridoma cell lines.
  • the host animal is inoculated intraperitoneally, intramuscularly, orally, subcutaneously, intraplantar, and/or intradermally with an amount of immunogen, including as described herein.
  • an antibody (monoclonal or polyclonal) of interest may be sequenced and the polynucleotide sequence may then be cloned into a vector for expression or propagation.
  • the sequence encoding an antibody of interest may be maintained in a vector in a host cell and the host cell can then be expanded and frozen for future use.
  • the polynucleotide sequence may be used for genetic manipulation to "humanize” an antibody or to improve the affinity (affinity maturation), or other characteristics of the antibody.
  • the constant region may be engineered to resemble human constant regions to avoid immune response if an antibody is used in clinical trials and treatments in humans.
  • Fully human antibodies can be obtained by using commercially available mice that have been engineered to express specific human immunoglobulin proteins.
  • Transgenic animals that are designed to produce a more desirable (e.g., fully human antibodies) or more robust immune response may also be used for generation of humanized or human antibodies. Examples of such technology are XenomouseRTM from Amgen, Inc. (Fremont, CA) and HuMAb-MouseRTM and TC MouseTM from Medarex, Inc. (Princeton, NJ) or H2L2 mice from Harbour Antibodies BV (Holland).
  • antibodies may be made recombinantly by phage display or yeast technology. See, for example, U.S. Pat. Nos.
  • Antigen-binding fragments of an intact antibody can be prepared via routine methods.
  • F(ab')2 fragments can be produced by pepsin digestion of an antibody molecule, and Fab fragments that can be generated by reducing the disulfide bridges of F(ab')2 fragments.
  • Genetically engineered antibodies such as humanized antibodies, chimeric antibodies, single-chain antibodies, and bi-specific antibodies, can be produced via, e.g., conventional recombinant technology.
  • DNA encoding monoclonal antibodies specific to a target antigen can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the monoclonal antibodies).
  • the hybridoma cells serve as a preferred source of such DNA.
  • the DNA may be placed into one or more expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, human HEK293 cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
  • the DNA can then be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences, Morrison et al., (1984) Proc. Nat. Acad. Sci. 81:6851, or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide.
  • genetically engineered antibodies such as “chimeric” or “hybrid” antibodies; can be prepared that have the binding specificity of a target antigen.
  • a single-chain antibody can be prepared via recombinant technology by linking a nucleotide sequence coding for a heavy chain variable region and a nucleotide sequence coding for a light chain variable region. Preferably, a flexible linker is incorporated between the two variable regions.
  • techniques described for the production of single chain antibodies can be adapted to produce a phage or yeast scFv library and scFv clones specific to PRAME/HLA-A2 can be identified from the library following routine procedures. Positive clones can be subjected to further screening to identify those that have high PRAME/HLA-A2 binding affinity.
  • Antibodies obtained following a method known in the art and described herein can be characterized using methods well known in the art. For example, one method is to identify the epitope to which the antigen binds, or “epitope mapping.” There are many methods known in the art for mapping and characterizing the location of epitopes on proteins, including solving the crystal structure of an antibody-antigen complex, competition assays, gene fragment expression assays, and synthetic peptide-based assays, as described, for example, in Chapter 11 of Harlow and Lane, Using Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1999.
  • epitope mapping can be accomplished using H/D-Ex (hydrogen deuterium exchange) coupled with proteolysis and mass spectrometry.
  • epitope mapping can be used to determine the sequence to which an antibody binds.
  • the epitope can be a linear epitope, i.e., contained in a single stretch of amino acids, or a conformational epitope formed by a three- dimensional interaction of amino acids that may not necessarily be contained in a single stretch (primary structure linear sequence).
  • Peptides of varying lengths e.g., at least 4-6 amino acids long
  • the epitope to which an antibody binds can be determined in a systematic screening by using overlapping peptides derived from the target antigen sequence and determining binding by the antibody.
  • the gene fragment expression assays the open reading frame encoding the target antigen is fragmented either randomly or by specific genetic constructions and the reactivity of the expressed fragments of the antigen with an antibody to be tested is determined.
  • the gene fragments may, for example, be produced by PCR and then transcribed and translated into protein in vitro, in the presence of radioactive amino acids. The binding of an antibody to the radioactively labeled antigen fragments is then determined by immunoprecipitation and gel electrophoresis.
  • Certain epitopes can also be identified by using large libraries of random peptide sequences displayed on the surface of phage particles (phage libraries). Alternatively, a defined library of overlapping peptide fragments can be tested for binding to the test antibody in simple binding assays. In an additional example, mutagenesis of an antigen binding domain, domain swapping experiments and alanine scanning mutagenesis can be performed to identify residues required, sufficient, and/or necessary for epitope binding. Alternatively, competition assays can be performed using other antibodies known to bind to the same antigen to determine whether an antibody binds to the same epitope as the other antibodies. Competition assays are well known to those of skill in the art.
  • an anti-PRAME/HLA-A2 antibody is prepared by recombinant technology as exemplified below.
  • Nucleic acids encoding the heavy and light chain of an anti-PRAME/HLA-A2 antibody as described herein can be cloned into one expression vector, each nucleotide sequence being in operable linkage to a suitable promoter.
  • each of the nucleotide sequences encoding the heavy chain and light chain is in operable linkage to a distinct promoter.
  • the nucleotide sequences encoding the heavy chain and the light chain can be in operable linkage with a single promoter, such that both heavy and light chains are expressed from the same promoter.
  • an internal ribosomal entry site IRS
  • the nucleotide sequences encoding the two chains of an antibody are cloned into two vectors, which can be introduced into the same or different cells.
  • the two chains are expressed in different cells, each of them can be isolated from the host cells expressing such and the isolated heavy chains and light chains can be mixed and incubated under suitable conditions allowing for the formation of an antibody.
  • a nucleic acid sequence encoding one or all chains of an antibody can be cloned into a suitable expression vector in operable linkage with a suitable promoter using methods known in the art.
  • the nucleotide sequence and vector can be contacted, under suitable conditions, with a restriction enzyme to create complementary ends on each molecule that can pair with each other and be joined together with a ligase.
  • synthetic nucleic acid linkers can be ligated to the termini of a gene. These synthetic linkers contain nucleic acid sequences that correspond to a particular restriction site in the vector. The selection of expression vectors/promoters would depend on the type of host cells for use in producing the antibodies.
  • a variety of promoters can be used for expression of the antibodies described herein, including, but not limited to, cytomegalovirus (CMV) intermediate early promoter, a viral LTR such as the Rous sarcoma virus LTR, HIV-LTR, HTLV-1 LTR, the simian virus 40 (SV40) early promoter, E. coli lac UV promoter, and the herpes simplex tk virus promoter.
  • CMV cytomegalovirus
  • a viral LTR such as the Rous sarcoma virus LTR, HIV-LTR, HTLV-1 LTR
  • SV40 simian virus 40
  • E. coli lac UV promoter E. coli lac UV promoter
  • herpes simplex tk virus promoter E. coli lac UV promoter
  • tetR tetracycline repressor
  • Other systems include FK506 dimer, VP 16 or p65 using astradiol, RU486, diphenol murislerone, or rapamycin. Inducible systems are available from Invitrogen, Clontech and Ariad, among others.
  • Regulatable promoters that include a repressor with the operon can be used.
  • the lac repressor from E. coli can function as a transcriptional modulator to regulate transcription from lac operator-bearing mammalian cell promoters [[M. Brown et al., Cell, 49:603-612 (1987)]]; Gossen and Bujard (1992); [[M. Gossen et al., Natl. Acad. Sci.
  • tetracycline repressor tetR
  • VP 16 transcription activator
  • tetO bearing minimal promoter derived from the human cytomegalovirus (hCMV) promoter to create a tetR-tet operator system to control gene expression in mammalian cells.
  • hCMV human cytomegalovirus
  • a tetracycline inducible switch is used.
  • the tetracycline repressor (tetR) alone, rather than the tetR-mammalian cell transcription factor fusion derivatives can function as potent trans-modulator to regulate gene expression in mammalian cells when the tetracycline operator is properly positioned downstream for the TATA element of the CMVIE promoter (Yao et al., Human Gene Therapy).
  • tetracycline inducible switch is that it does not require the use of a tetracycline repressor-mammalian cells transactivator or repressor fusion protein, which in some instances can be toxic to cells (Gossen 5 et al., Natl. Acad. Sci. USA, 89:5547-5551 (1992); Shockett et al., Proc. Natl. Acad. Sci. USA, 92:6522-6526 (1995)), to achieve its regulatable effects.
  • the vector can contain, for example, some or all of the following: a selectable marker gene, such as the neomycin gene for selection of stable or transient transfectants in mammalian cells; enhancer/promoter sequences from the immediate early gene of human CMV for high levels of transcription; transcription termination and RNA processing signals from SV40 for mRNA stability; SV40 polyoma origins of replication and ColEl for proper episomal replication; internal ribosome binding sites (IRESes), versatile multiple cloning sites; and T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA.
  • a selectable marker gene such as the neomycin gene for selection of stable or transient transfectants in mammalian cells
  • enhancer/promoter sequences from the immediate early gene of human CMV for high levels of transcription
  • transcription termination and RNA processing signals from SV40 for mRNA stability SV40 polyoma origins of replication and ColEl for proper episomal replication
  • One or more vectors comprising nucleic acids encoding any of the antibodies (e.g., the nucleic acid coding sequence listed in Table 3) may be introduced into suitable host cells for producing the antibodies.
  • Non-limiting examples of the host cells include Chinese hamster ovary (CHO) cells, dhfr- CHO cell, human embryonic kidney (HEK)-293 cells, verda reno (VERO) cells, nonsecreting null (NSO) cells, human embryonic retinal (PER.C6) cells, Sp2/0 cells, baby hamster kidney (BHK) cells, Madin- Darby Canine Kidney (MDCK) cells, Madin-Darby Bovine Kidney (MDBK) cells, and monkey kidney CV1 line transformed by SV40 (COS) cells.
  • the host cell expressing the anti-PRAME/HLA-A2 antibodies are CHO cells.
  • the host cells can be cultured under suitable conditions for expression of an antibody or any polypeptide chain thereof. Such antibodies or polypeptide chains thereof can be recovered by the cultured cells (e.g., from the cells or the culture supernatant) via a conventional method, e.g., affinity purification. If necessary, polypeptide chains of an antibody can be incubated under suitable conditions for a suitable period of time allowing for production of the antibody.
  • the host cell comprises the nucleic acid encoding the heavy chain of the anti- PRAME/HLA-A2 antibody.
  • the host cell comprises the nucleic acid encoding the light chain of the anti-PRAME/HLA-A2 antibody.
  • the host cell comprises the nucleic acid encoding the heavy chain and the nucleic acid encoding the light chain.
  • methods for preparing an antibody described herein involve a recombinant expression vector that encodes both the heavy chain and the light chain of an anti-PRAME/HLA-A2 antibody, as also described herein.
  • the recombinant expression vector can be introduced into a suitable host cell (e.g., a dhfr- CHO cell) by a conventional method, e.g., calcium phosphate mediated transfection.
  • a suitable host cell e.g., a dhfr- CHO cell
  • Positive transformant host cells can be selected and cultured under suitable conditions allowing for the expression of the two polypeptide chains that form an antibody, which can be recovered from the cells or from the culture medium.
  • the two chains recovered from the host cells can be incubated under suitable conditions allowing for the formation of an antibody.
  • two recombinant expression vectors are provided, one encoding the heavy chain of the anti-PRAME/HLA-A2 antibody and the other encoding the light chain of the anti-PRAME/HLA-A2 antibody.
  • Both of the two recombinant expression vectors can be introduced into a suitable host cell (e.g., dhfr- CHO cell) by a conventional method, e.g., calcium phosphate-mediated transfection.
  • each of the expression vectors can be introduced into suitable host cells. Positive transformants can be selected and cultured under suitable conditions allowing for the expression of the polypeptide chains of an antibody.
  • an antibody produced therein can be recovered from the host cells or from the culture medium.
  • the polypeptide chains can be recovered from the host cells or from the culture medium and then incubated under suitable conditions allowing for formation of an antibody.
  • each of them can be recovered from the corresponding host cells or from the corresponding culture media. The two polypeptide chains can then be incubated under suitable conditions for formation of an antibody.
  • Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells and recovery of the antibodies from the culture medium.
  • some antibodies can be isolated by affinity chromatography with a Protein A or Protein G coupled matrix.
  • nucleic acids encoding the heavy chain, the light chain, or both of an anti-PRAME/HLA-A2 antibody as described herein e.g., as provided in Table 3
  • vectors e.g., expression vectors
  • host cells comprising the vectors
  • an antibody provided herein may be produced by expressing a polypeptide comprising the heavy chain and light chain CDRs of any antibody provided herein.
  • a single polypeptide comprises a HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 of any antibody provided herein.
  • a polypeptide e.g., a first polypeptide
  • a polypeptide comprises a HC CDR1, HC CDR2, and HC CDR3 of any antibody provided herein
  • a polypeptide e.g., a second polypeptide
  • the first polypeptide and the second polypeptide form a polypeptide complex.
  • a polypeptide complex may be further modified (e.g., by protein folding, by post-translational modifications, or any other modification either singly or in combination).
  • a polypeptide complex is any antibody provided herein.
  • Table 3 Nucleic acids Sequences encoding the VH/VL of anti-PRAME/HLA-A2 binders listed in Table 1.
  • the present disclosure provides an isolated nucleic acid comprising a sequence at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 43.
  • the present disclosure provides an isolated nucleic acid comprising a sequence at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to nucleic acid #1 in row 67; nucleic acid #2 in row 68, nucleic acid #3 in row 69, nucleic acid #4 in row 70, nucleic acid #5 in row 71, nucleic acid #6 in row 72, nucleic acid #7 in row 73, nucleic acid #8 in row 74, nucleic acid #9 in row 75, nucleic acid #10 in row 76, nucleic acid #11 in row 77, nucleic acid #12 in row 78, nucleic acid #13 in row 79, nucleic acid #14 in row 80, nucleic acid #15 in row 81, nucleic acid #16 in row 82, nucleic acid #17 in row
  • the present disclosure provides an isolated nucleic acid comprising a sequence at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 43 and a sequence at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to nucleic acid #1 in row 67; nucleic acid #2 in row 68, nucleic acid #3 in row 69, nucleic acid #4 in row 70, nucleic acid #5 in row 71, nucleic acid #6 in row 72, nucleic acid #7 in row 73, nucleic acid #8 in row 74, nucleic acid #9 in row 75, nucleic acid #10 in row 76, nucleic acid
  • the present disclosure provides an isolated nucleic acid comprising a sequence at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to nucleic acid #1 in row 67; nucleic acid #2 in row 68, nucleic acid #3 in row 69, nucleic acid #4 in row 70, nucleic acid #5 in row 71, nucleic acid #6 in row 72, nucleic acid #7 in row 73, nucleic acid #8 in row 74, nucleic acid #9 in row 75, nucleic acid #10 in row 76, nucleic acid #11 in row 77, nucleic acid #12 in row 78, nucleic acid #13 in row 79, nucleic acid #14 in row 80, nucleic acid #15 in row 81, nucleic acid #16 in row 82, nucleic acid
  • the present disclosure provides an isolated nucleic acid comprising a sequence at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to nucleic acid #1 in row 67; nucleic acid #2 in row 68, nucleic acid #3 in row 69, nucleic acid #4 in row 70, nucleic acid #5 in row 71, nucleic acid #6 in row 72, nucleic acid #7 in row 73, nucleic acid #8 in row 74, nucleic acid #9 in row 75, nucleic acid #10 in row 76, nucleic acid #11 in row 77, nucleic acid #12 in row 78, nucleic acid #13 in row 79, nucleic acid #14 in row 80, nucleic acid #15 in row 81, nucleic acid #16 in row 82, nucleic acid #17 in row
  • the present disclosure provides an isolated nucleic acid comprising a sequence at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to nucleic acid #1 in row 67; nucleic acid #2 in row 68, nucleic acid #3 in row 69, nucleic acid #4 in row 70, nucleic acid #5 in row 71, nucleic acid #6 in row 72, nucleic acid #7 in row 73, nucleic acid #8 in row 74, nucleic acid #9 in row 75, nucleic acid #10 in row 76, nucleic acid #11 in row 77, nucleic acid #12 in row 78, nucleic acid #13 in row 79, nucleic acid #14 in row 80, nucleic acid #15 in row 81, nucleic acid #16 in row 82, nucleic acid #17 in row
  • the present disclosure provides methods for producing an antibody, the methods comprising culturing the recombinant cells under conditions suitable for expression of the antibody from the expression vector by the recombinant cell.
  • Recombinant cells expressing an antibody can be cultured in any suitable condition known in the art.
  • the method further comprises isolating the antibody from the culture media in which the cell or cells were cultured using any suitable known methods in the art.
  • the antibodies e.g., anti-PRAME/HLA-A2 antibody
  • a pharmaceutically acceptable carrier excipient
  • “Acceptable” means that the carrier must be compatible with the active ingredient of the composition (and preferably, capable of stabilizing the active ingredient) and not deleterious to the subject to be treated.
  • the anti-PRAME/HLA-A2 antibody containing pharmaceutical composition disclosed herein may further comprise a suitable buffer agent.
  • a buffer agent is a weak acid or base used to maintain the pH of a solution near a chosen value after the addition of another acid or base.
  • the buffer agent disclosed herein can be a buffer agent capable of maintaining physiological pH despite changes in carbon dioxide concentration (produced by cellular respiration).
  • Exemplary buffer agents include, but are not limited to, a HEPES (4-(2-hydroxyethyl)-l -piperazineethanesulfonic acid) buffer, Dulbecco's phosphate- buffered saline (DPBS) buffer, or Phosphate-buffered Saline (PBS) buffer.
  • DPBS Dulbecco's phosphate- buffered saline
  • PBS Phosphate-buffered Saline
  • Such buffers may comprise disodium hydrogen phosphate and sodium chloride, or potassium dihydrogen phosphate and potassium chlor
  • the buffer agent in the pharmaceutical composition described herein may maintain a pH value of about 5-8.
  • the pH of the pharmaceutical composition can be about 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0.
  • the pharmaceutical composition may have a pH value lower than 7, for example, about 7, 6.8, 6.5, 6.3, 6, 5.8, 5.5, 5.3, or 5.
  • the pharmaceutical composition described herein comprises one or more suitable salts.
  • a salt is an ionic compound that can be formed by the neutralization reaction of an acid and a base. (Skoog, D.A; West, D.M.; Holler, J.F.; Crouch, S.R. (2004). “Chapters 14-16”. Fundamentals of Analytical Chemistry (8th ed.)). Salts are composed of related numbers of cations (positively charged ions) and anions (negative ions) so that the product is electrically neutral (without a net charge).
  • the pharmaceutical compositions can comprise pharmaceutically acceptable carriers, excipients, or stabilizers in the form of lyophilized formulations or aqueous solutions.
  • the pharmaceutical composition can be formulated for intravenous injection.
  • the pharmaceutical composition can be formulated for subcutaneous injection.
  • the pharmaceutical compositions to be used for in vivo administration must be sterile. This is readily accomplished by, for example, filtration through sterile filtration membranes.
  • Therapeutic antibody compositions are generally placed into a container having a sterile access port, for example, an intravenous or subcutaneous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
  • PRAME is a tumor-associated antigen, and its expression has been observed in many types of cancer, including but not limited to melanoma, leukemia, head and neck cancer, renal cell cancer, breast cancer, and lung cancer.
  • PRAME is an intracellular protein thought to act as a repressor of retinoic acid signaling and is typically only expressed in germline cells (e.g., sperm cells).
  • germline cells e.g., sperm cells
  • PRAME is processed by the proteosome such that fragments of PRAME are expressed on MHC molecules on the cell surface, forming PRAME/HLA-A2 complexes on human cancer cells.
  • the antibodies provided by the present disclosure bind PRAME/HLA-A2 with high specificity.
  • the anti-PRAME/HLA-A2 antibody contains a binding site that specifically binds to PRAME/HLA-A2, and a second binding site.
  • the second binding site binds specifically to a T cell antigen.
  • the T cell antigen is a CD3 complex.
  • the second binding site specifically binds to CD38 of the CD3 complex.
  • the second binding site specifically binds to CD3e of the CD3 complex.
  • the second binding site specifically binds to CD3yof the CD3 complex.
  • the second binding site specifically binds to a CD38/E heterodimer of the CD3 complex.
  • the second binding site specifically binds to a CD3e/y heterodimer of the CD3 complex.
  • the anti-PRAME/HLA-A2 antibodies can direct cytotoxic T cells against PRAME/HLA- A2 expressing cancer cells, inducing death in those cancer cells.
  • the disclosure features a method for treating cancer (e.g., cancer expressing PRAME), the method comprising administering to a subject in need an effective amount of a therapeutic agent, wherein the therapeutic agent is or comprises: (i) any one or more of the antibodies or antigen-binding fragments thereof described herein (including conjugates), (ii) any one or more of the fusion proteins described herein, (iii) any one or more of the bispecific or multispecific polypeptides described herein; (iv) any one or more of the nucleic acids described herein; (v) any one or more of the expression vectors described herein; (vi) any one or more of the recombinant cells described herein; (vii) any one or more of the isolated polypeptides described herein; and/or (viii) any one or more of the pharmaceutical compositions described herein.
  • the therapeutic agent is or comprises: (i) any one or more of the antibodies or antigen-binding fragments thereof described herein (including conjugates), (ii) any one or more
  • the therapeutic agent can be administered through injection by intravenous, intraperitoneal, intracerebral (intra-parenchymal), intracerebroventricular, intramuscular, subcutaneously, intra-ocular, intraarterial, intraportal, or intralesional routes; by sustained release systems or by implantation devices.
  • the compositions can be administered by bolus injection or continuously by infusion, or by implantation device.
  • an amount of an antibody e.g., anti-PRAME/HLA-A2 antibody
  • Effective amounts vary, as recognized by those skilled in the art, depending on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size, gender and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner.
  • the particular dosage regimen, i.e., dose, timing, and repetition, used in the method described herein will depend on the particular subject and that subject's medical history, as discussed herein.
  • Empirical considerations such as time to maximum effect, the half-life, and/or time above a specific concentration generally will contribute to the determination of the dosage.
  • antibodies that are compatible with the human immune system such as humanized antibodies or fully human antibodies, may be used to prolong the half-life of an antibody and to prevent the antibody being attacked by the host's immune system.
  • Other reasons for dose-adjusting include differences in pharmacokinetics or pharmacodynamic response driven by sex, age, individual response, polymorphisms on an antibody target and/or receptors involved in antibody clearance.
  • Frequency of administration may be determined and adjusted over the course of therapy, and is generally, but not necessarily, based on treatment and/or suppression and/or amelioration and/or delay of a target disease/disorder.
  • sustained continuous release formulations of an antibody may be appropriate.
  • Various formulations and devices for achieving sustained release are known in the art.
  • Dosing frequencies may vary in accordance with the claimed methods.
  • a composition may be administered once.
  • a composition will be administered on multiple occasions.
  • dosing frequency is every week, every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, or every 10 weeks; or once every month, every 2 months, or every 3 months, or longer.
  • a composition will be administered daily, biweekly, weekly, bimonthly, monthly, or at any time interval that provides suitable (e.g., maximal) efficacy while minimizing safety risks to the subject. Generally, the efficacy and the treatment and safety risks may be monitored throughout the course of treatment.
  • a subject may be administered a composition provided herein (e.g., an anti-PRAME/HLA-A2 antibody) at one or more intervals during a set period of time.
  • periods of time during which a subject is administered a composition at one or more intervals may be separated by periods of time in which the subject is not administered the composition.
  • the relative durations of respective periods of time may depend on the subject’s response to treatment or severity of disease or both and/or may be determined based on the judgment of a treating physician.
  • an antibody can be administered parenterally.
  • a parenterally administered composition may be administered by subcutaneous, intracutaneous, intravenous, intraperitoneal, intratumor, intramuscular, intraarticular, intraarterial, or infusion techniques.
  • it can be administered to the subject via injectable depot routes of administration such as using 1-, 3-, or 6-month depot injectable or biodegradable materials and methods.
  • an antibody e.g., an anti-PRAME/HLA-A2 antibody
  • an antibody e.g., an anti-PRAME/HLA-A2 antibody
  • an antibody is administered subcutaneously.
  • water soluble antibodies can be administered by the drip method, whereby a pharmaceutical formulation containing an antibody and a physiologically acceptable excipient is infused.
  • Physiologically acceptable excipients may include, for example, 5% dextrose, 0.9% saline. Ringer’s solution or other suitable excipients.
  • Other injectable compositions may contain various carriers such as vegetable oils, dimethylactamide, dimethyformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols (glycerol, propylene glycol, liquid polyethylene glycol, and the like).
  • preparations e.g., a sterile formulation of a suitable soluble salt form of an antibody
  • a pharmaceutical excipient such as Water-for- Injection, 0.9% saline, or 5% glucose solution.
  • an antibody is administered via site- specific or targeted local delivery techniques.
  • site- specific or targeted local delivery techniques include various implantable depot sources of an antibody or local delivery catheters, such as infusion catheters, an indwelling catheter, or a needle catheter, synthetic grafts, adventitial wraps, shunts and stents or other implantable devices, site specific carriers, direct injection, or direct application. See, e.g., PCT Publication No. WO 00/53211 and U.S. Pat. No. 5,981,568.
  • more than one antibody, or a combination of an antibody and another suitable therapeutic agent may be administered to a subject in need of the treatment.
  • An antibody can also be used in conjunction with other agents that serve to enhance and/or complement the effectiveness of the agents.
  • Treatment efficacy for a target disease/disorder can be assessed by methods well-known in the art.
  • the anti-PRAME/HLA-A2 antibody and treatment methods involving such as described in the present disclosure may be utilized in combination with other types of therapy for the target disease or disorder disclosed herein.
  • an antibody composition and a therapeutic agent may be given either simultaneously or sequentially. Examples include chemotherapy, immune therapy, surgery, radiation, gene therapy, and so forth, or antiinfection therapy.
  • Such therapies can be administered simultaneously or sequentially (in any order) with the treatment according to the present disclosure.
  • the combination therapy can include the anti-PRAME/HLA-A2 antibody and pharmaceutical composition described herein, co-formulated with and/or coadministered with, at least one additional therapeutic agent.
  • Such combination therapies may advantageously utilize lower dosages of the administered therapeutic agents, thus preventing possible toxicities or complications associated with the various monotherapies.
  • combination therapies may comprise an anti- PRAME/HLA-A2 antibody provided herein (antibody #1, antibody #2, antibody #3, antibody #4, antibody #5, antibody #6, antibody #7, antibody #8, antibody #9, antibody #10, antibody #11, antibody #12, antibody #13, antibody #14, antibody #15, antibody #16, antibody #17, antibody #18, antibody #19, antibody #20, antibody #21, antibody #22, antibody #23, antibody #24, antibody #25, antibody #26, antibody #27, antibody #28, antibody #29, antibody #30, antibody #31, antibody #32, antibody #33) in combination with a therapeutic agent.
  • a therapeutic agent is a chemotherapeutic agent.
  • the antibodies described herein are conjugated directly or indirectly to one or more molecular pay loads or labels.
  • antibodies described herein are conjugated to molecular payload, e.g., a molecular payload providing a therapeutic benefit for a subject, e.g., an antibody-drug conjugate (ADC).
  • ADC antibody-drug conjugate
  • methods are provided for delivering molecular payloads to a subject for therapeutic purposes.
  • the molecular pay load may be a small molecule, protein, nucleic acid, oligonucleotide, or any molecular entity capable of modulating the activity or function of a gene, protein, and/or nucleic acid, e.g., in a cell.
  • the molecular payload is a cytotoxic agent or a chemotherapeutic agent.
  • Any of the anti-PRAME/HLA-A2 antibodies disclosed herein can also be used for detecting presence of PRAME/HLA-A2 in vitro or in vivo.
  • Results obtained from such detection methods can be used for diagnostic purposes (e.g., diagnosing diseases associated with PRAME/HLA-A2) or for scientific research purposes (e.g., identifying new PRAME/HLA-A2- secreting cell types, studying bioactivity and/or regulation of secreted PRAME/HLA-A2).
  • an anti-PRAME/HLA-A2 antibody as described herein may be conjugated with a detectable label (e.g., an imaging agent such as a contrast agent) for detecting presence of PRAME/HLA-A2 (e.g., soluble PRAME/HLA-A2), either in vivo or in vitro.
  • a detectable label e.g., an imaging agent such as a contrast agent
  • an anti-PRAME/HLA-A2 antibody as described herein can be attached to a detectable label, which is a compound that is capable of releasing a detectable signal, either directly or indirectly, such that the aptamer can be detected, measured, and/or qualified, in vitro or in vivo.
  • detectable labels are intended to include, but are not limited to, fluorescent labels, chemiluminescent labels, colorimetric labels, enzymatic markers, radioactive isotopes, and affinity tags such as biotin.
  • Such labels can be conjugated to the aptamer, directly or indirectly, by conventional methods.
  • the detectable label is an agent suitable for detecting PRAME/HLA-A2 expressing cells in vitro, which can be a radioactive molecule, a radiopharmaceutical, or an iron oxide particle.
  • Radioactive molecules suitable for in vivo imaging include, but are not limited to, 122 I, 123 I, 124 I, 125 I, 131 I, 18 F, 75 Br, 76 Br, 77 Br, 211 At, 225 Ac, 177 LU, 153 Sm, 186 Re, 188 Re, 67 Cu, 213 Bi, 212 Bi, 212 Pb, and 67 Ga.
  • radiopharmaceuticals suitable for in vivo imaging include in In Oxyquinoline, 131 I Sodium iodide, “mTc Mebrofenin, and “mTc Red Blood Cells, 123 I Sodium iodide, "mTc Exametazime, "mTc Macroaggregate Albumin, “mTc Medronate, “mTc Mertiatide, “mTc Oxidronate, “mTc Pentetate, “mTc Pertechnetate, “mTc Sestamibi, “mTc Sulfur Colloid, “mTc Tetrofosmin, Thallium-201, or Xenon- 133.
  • the reporting agent can also be a dye, e.g., a fluorophore, which is useful in detecting a disease mediated by PRAME/HLA-A2 expressing cells in tissue samples.
  • a dye e.g., a fluorophore
  • an anti- PRAME/HLA-A2 antibody can be brought in contact with a sample suspected of containing PRAME/HLA-A2, e.g., PRAME/HLA-A2 expressing cells or soluble PRAME/HLA-A2 in disease microenvironment.
  • An antibody and the sample may be incubated under suitable conditions for a suitable period to allow for binding of the antibody to the PRAME/HLA-A2 antigen.
  • an interaction can then be detected via routine methods, e.g., ELISA, histological staining, or FACS.
  • a suitable amount of anti- PRAME/HLA-A2 antibodies, conjugated with a label can be administered to a subject in need of the examination. Presence of the labeled antibody can be detected based on the signal released from the label by routine methods.
  • an anti-PRAME/HLA-A2 antibody can be used to study bioactivity of PRAME/HLA-A2, detect the presence of PRAME/HLA-A2 intracellularly, and/or regulating the effect of PRAME/HLA-A2.
  • a suitable amount of anti-PRAME/HLA-A2 can be brought in contact with a sample (e.g., a new cell type that is not previously identified as PRAME/HLA-A2 producing cells) suspected of producing PRAME/HLA-A2.
  • the cells are permeabilized prior to contacting the anti- PRAME/HLA-A2 antibody.
  • An antibody and the sample may be incubated under suitable conditions for a suitable period to allow for binding of the antibody to the PRAME/HLA-A2 antigen.
  • Such an interaction can then be detected via routine methods, e.g., ELISA, histological staining, or FACS.
  • kits for the therapeutic or diagnostic applications as disclosed herein can include one or more containers comprising an anti-PRAME/HLA-A2 antibody, e.g., any of those described herein.
  • the kit can comprise instructions for use in accordance with any of the methods described herein.
  • the included instructions can comprise a description of administration of the anti-PRAME/HLA-A2 antibody to treat, delay the onset, or alleviate a target disease as those described herein.
  • the kit may further comprise a description of selecting an individual suitable for treatment based on identifying whether that individual has the target disease.
  • the instructions comprise a description of administering an antibody to an individual at risk of the target disease.
  • the instructions relating to the use of an anti-PRAME/HLA-A2 antibody generally include information as to dosage, dosing schedule, and route of administration for the intended treatment.
  • the containers may be unit doses, bulk packages (e.g., multi-dose packages) or sub-unit doses.
  • Instructions supplied in the kits of the invention are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.
  • the label or package insert indicates that the composition is used for treating, delaying the onset, and/or alleviating a disease or disorder. Instructions may be provided for practicing any of the methods described herein.
  • kits of this invention are in suitable packaging.
  • suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like.
  • kits for use in combination with a specific device such as an infusion device, such as a minipump.
  • a kit may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
  • the container may also have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
  • At least one active agent in the composition is an anti- PRAME/HLA-A2 antibody as those described herein.
  • Kits may optionally provide additional components such as buffers and interpretive information.
  • the kit comprises a container and a label or package insert(s) on or associated with the container.
  • the invention provides articles of manufacture comprising contents of the kits described above.
  • kits for use in detecting PRAME/HLA-A2 in a sample may comprise any of the anti-PRAME/HLA-A2 antibodies described herein.
  • the anti-PRAME/HLA-A2 antibody can be conjugated with a detectable label as those described herein. Conjugated or attached can include covalent or noncovalent bonding as well as other forms of association, such as entrapment, e.g., of one entity on or within the other, or of either or both entities on or within a third entity, such as a micelle.
  • the kit may comprise a secondary antibody capable of binding to anti-PRAME/HLA-A2 antibody.
  • the kit may further comprise instructions for using the anti-PRAME/HLA-A2 antibody for detecting PRAME/HLA-A2.
  • Example 1 Generation of candidate anti-PRAME/HLA-A2 candidates.
  • mice which produce human antibodies, were immunized subcutaneously with PRAME/HLA-A2 complex carrying an His-tag on a weekly basis. After a total of 5 injections, mice were sacrificed and their splenocytes were used for antigen- specific B cell isolation by fluorescence-assisted cell sorting and to build immune- phage libraries.
  • mice sera were prepared and used in an enzyme-linked immunosorbent assay (ELISA) against PRAME/HLA-A2 protein complex. Briefly, PRAME/HLA-A2 protein complex was coated on an ELISA plate. Plates were blocked with 3% BSA buffer for Jackpot and after PBS wash, sera dilutions were transferred to the ELISA plate and incubated for Jackpot. After PBS wash, a secondary antibody specific for mouse IgG was added to the ELISA plate for 1 hr. After PBS wash, TMB solution was added, and reaction was then stopped using sulfuric acid.
  • ELISA enzyme-linked immunosorbent assay
  • Splenic B cells were stained with a panel of surface marker antibodies and a fluorescently labeled PRAME/HLA-A2 biotinylated using previously published protocols (Tiller et al., J Immunol Methods, 2009. 350( 1-2): 183- 193).
  • PRAME/HLA-A2- specific B cells were sorted one cell per well in lysis buffer and were frozen.
  • Heavy and light chain sequences were retrieved using previously published protocols (Tiller et al., J Immunol Methods, 2009. 350(1-2): 183- 193; Tiller et al., J Immunol Methods, 2008. 329(1-2): 112-124). After sequencing, heavy and light chain genes were cloned into expression vectors.
  • Example 3 Cell Binding Assay of anti-PRAME/HLA-A2 candidates.
  • T2 cells at a concentration of 10 6 cells/mL were incubated with 50 pM peptide of interest or control peptide overnight in serum-free RPMI medium supplemented with 5 mg/mL human b2-microglobulin (Sigma, St. Louis, MO, USA). After the incubation, T2 cells were washed with PBS. In the antibody-cell binding experiment, the antibodies were tested at a concentration from 100 nM to 0.6 pM (a serial 3-fold dilution) for binding on the prepared T2 cells for 45 min at 4°C. Cells were then incubated with the secondary antibody R- Phycoerythrin AffiniPure Goat Anti-Human IgG (Jackson Immunoresearch 109-115-098).
  • the data was acquired on FACSCanto II (BD) or on Intellicyt iQue3. Median fluorescence intensities (MFI) were plotted against the concentrations of the antibodies. EC50 was derived from fitting to 4 parameter dose-response curve.
  • Example 4 Polyspecificity testing of anti-PRAME/HLA-A2 candidates.
  • Phages were loaded with either PRAME peptides (VLDGLDVLL (SEQ ID NO: 17) or SLLQHLIGL (SEQ ID NO: 19) or one of approximately 100 non-target peptides complexed with HLA-A2 (e.g., TGF-P, insulin, p53, and others) (FIGs. 2A-2B, 3). Binding strength of candidate anti-PRAME/HLA-A2 antibodies was measured, and binders that strongly and specifically bind the SLLQHLIGL (SEQ ID NO: 19) PRAME peptide in complex with HLA- A2 were identified (FIG. 3). The EC50 and KD of each candidate antibody is shown in Table 5 below.
  • MFI mean fluorescence intensity
  • Example 5 Identification of critical epitope residues using X-scanning.
  • sc-pHLA single-chain peptide/HLA
  • gBlock pools were synthesized by IDT and then cloned into the phagemid DNA to display sc-pHLA molecules on the surface of phage particles. IxlO 11 phage particles were produced for each panning round and three rounds of panning were performed.
  • a phage pool was incubated with TCRm coated streptavidin beads for 1 hour at room temperature and then washed with 6 times of PBST and 3 times of PBS to remove unbound phage particles. Bead bound phage particles were eluted with 100 mM Triethylamine (TEA) and then infected TGI cells for the later phagemid DNA extraction. Two specific primers were used to amplify the peptide region of collected phagemid DNA to generate amplicons for next-generation sequencing (NGS). The NGS data was then processed by the bioinformatic team to plot the heatmap and seqlogo graphs based on the frequency of each peptide in the 3 panning rounds (FIGs. 5-7). For additional methods, see Coles et al., J Immunol, 2020. 204(7): 1943-1953. [000304] Example 6. Functional testing of anti-PRAME/HLA-A2 candidate activity.
  • Bispecific antibodies containing anti-PRAME/HLA-A2 binding arms engineered in an anti-CD3-containing format will be tested in co-culture assays using Jurkat NFAT- luciferase reporter cells co-cultured with T2 accessory cells pulsed with target (PRAME- SLL) or irrelevant (PRAME-VLD) peptides.
  • Human PBMCs from several unique HLA- A*02.01 donors will be acquired and CD8+ T cells will be isolated and cryopreserved. These CD8+ T cells will be co-cultured with a variety of human tumor cell lines, including controls which are PRAME-SLL positive or negative, and controls which are HLA-A*02.01 positive or negative.
  • Functional readouts will include: target cell cytotoxicity (measured by live-cell imaging and/or flow cytometry), cytokine production from the co-culture supernatant (measured by multiplexed electrochemiluminescence ELISA). Additionally, bulk PBMCs from several unique HLA-A*02.01 donors will be co-cultured with a variety of human tumor cell lines, including controls which are PRAME-SLL positive or negative, and controls which are HLA-A*02.01 positive or negative. Functional readouts will include: target cell cytotoxicity (measured by flow cytometry), immunoprofiling including T-cell activation/exhaustion (measured by flow cytometry).
  • Example 7 Toxicity and Tissue reactivity of anti-PRAME/HLA-A2 candidate activity.
  • Bispecific antibodies containing anti-PRAME/HLA-A2 binding arms engineered in an anti-CD3-containing format will be incubated with whole blood from several unique human donors to quantify whether there is CD3-dependent cytokine release, indicative of potential clinical toxicities. Additionally, a variety of normal human tissues and normal human cells lines (i.e., not cancer cell lines) will be procured and bispecific antibodies will be assessed for off-target binding to any proteins or HLA/peptide complexes present in normal healthy human tissue.
  • articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context.
  • the invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process.
  • the invention includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.
  • the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim.
  • any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim.
  • elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group.
  • the invention, or aspects of the invention is/are referred to as comprising particular elements and/or features, certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements and/or features. For purposes of simplicity, those embodiments have not been specifically set forth in haec verba herein.
  • a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
  • the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements.
  • This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified.
  • “at least one of A and B” can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

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Abstract

Certains aspects de l'invention concernent une composition (par exemple, des anticorps anti-PRAME/HLA-A2, et/ou des anticorps bispécifiques anti-PRAME/A2xcellule T) et des méthodes d'utilisation de celle-ci dans le traitement du cancer (par exemple, le cancer exprimant PRAME).
PCT/US2025/035306 2024-06-25 2025-06-25 Anticorps anti-prame/hla-a2 et leurs utilisations Pending WO2026006492A2 (fr)

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