Antigen-binding protein that specifically binds to PRAME

Abstract

The present invention relates to an antigen binding protein for an antigen derived from PRAME protein. The present invention provides in particular an antigen binding protein specific for the tumor expressed antigen PRAME, which comprises or consists of SEQ ID NO: 50 and is present in a complex with a major histocompatibility complex (MHC) protein. The antigen binding protein of the present invention in particular comprises a novel engineered T cell receptor (TCR) complementarity determining region (CDR) that specifically binds to said PRAME peptide. The antigen binding protein of the present invention is used for the diagnosis, treatment and prevention of PRAME-expressing cancerous diseases. Nucleic acids encoding the antigen binding protein of the present invention, vectors comprising said nucleic acids, recombinant cells expressing said antigen binding protein, and pharmaceutical compositions comprising the antigen binding protein of the present invention are further provided.

Description

[Technical Field]

[0001] The present invention relates to an antigen-binding protein for a PRAME protein-derived antigen. In particular, the present invention provides an antigen-binding protein specific to the PRAME antigen expressed by a tumor, wherein the tumor antigen comprises or consists of SEQ ID NO: 50 and exists in complex with a major histocompatibility complex (MHC) protein. The antigen-binding protein of the present invention particularly comprises a novel, engineered T-cell receptor (TCR) complementarity-determining region (CDR) that specifically binds to the PRAME peptide. The antigen-binding protein of the present invention is used in the diagnosis, treatment, and prevention of PRAME-expressing cancers. Further provided are nucleic acids encoding the antigen-binding protein of the present invention, vectors containing the nucleic acid, recombinant cells expressing the antigen-binding protein, and pharmaceutical compositions containing the antigen-binding protein of the present invention. [Background technology]

[0002] PRAME stands for "Preferentially Expressed Antigen in Melanoma" and belongs to a family of germline-encoded antigens known as cancer-testis antigens. Cancer-testis antigens are targets for immunotherapy interventions. PRAME is expressed in many solid tumors, as well as leukemia and lymphoma. The peptide SLLQHLIGL (SEQ ID NO: 50), also known as PRAME-004, corresponds to amino acids 425-433 of the full-length PRAME protein (SEQ ID NO: 328), and this peptide is presented on the cell surface in complex with MHC molecules (particularly HLA-A*02) (Kessler et al., J Exp Med. 2001 Jan 1;193(1):73-88). TCRs can bind to the peptide epitope presented by MHC molecules.

[0003] While progress is being made in the development of molecularly targeted drugs for cancer treatment, this field still requires the development of novel anticancer agents that specifically target molecules that are highly specific to cancer cells but not to normal tissue cells. PRAME-004 peptide is a target for T-cell-based immunotherapy because it is specifically expressed on tumors.

[0004] International Publication No. 2018 / 172533 discloses a TCR (e.g., TCR R11P3D3) that binds to the PRAME-004 peptide in complex with an MHC protein complex, and the use of said TCR in the diagnosis, treatment, and prevention of cancerous diseases that (over) express PRAME. However, this TCR does not have a CDR region designed to bind to the target antigen with high affinity.

[0005] Native TCRs typically have low affinity (K DTCRs bind to MHC-presenting antigens at affinities of 300 μM to 1 μM, and therefore, in contrast to viral exogenous antigens for which TCR binding affinity is well established in the range of 1 to 10 μM, binding to MHC-presenting cancer autoantigens with affinities greater than 10 μM is rarely observed (Aleksic et al. 2012, Eur J Immunol. 2012 Dec;42(12):3174-9). Part of the explanation for this phenomenon is as follows: T cells developing in the thymus are negatively selected on self-peptide-MHC ligands, and therefore, T cells with excessively high affinity for such self-peptide-MHCs are eliminated (tolerance induction). This low affinity of TCRs to cancer autoantigens may be one possible explanation for tumor immune evasion (Aleksic et al. 2012, Eur J Immunol. 2012 Dec;42(12):3174-9). Therefore, designing TCR variants that bind to cancer autoantigens with higher affinity for use as antigen recognition constructs in adoptive cell therapy (ACT) seems like a desirable strategy. Furthermore, designing high-affinity TCR variants that can be expressed as soluble proteins would be desirable for targeting cancer autoantigens with soluble therapeutic agents (i.e., using bispecific molecules) (Hickman et al. 2016, J Biomol Screen. 2016 Sep;21(8):769-85).

[0006] However, increasing the affinity of a TCR may also increase the risk of side effects. As mentioned above, in nature, high-affinity TCRs for tumor-associated antigens, which are self-proteins, are eliminated by thymic selection to avoid cross-reactivity and recognition of self-peptides present on normal tissues. Therefore, simply increasing the affinity of a TCR for its target sequence may also increase its affinity for non-cancer-specific, similar peptides, thus increasing the risk of cross-reactivity and undesirable cytotoxic effects on healthy tissues. This is not merely a theoretical risk, as has been painfully demonstrated with regard to engineered TCRs targeting MAGE-A3. In particular, previously published results have shown lethal toxicity in two patients who were injected with T cells engineered to express a MAGE-A3-targeting TCR that cross-reacts with a peptide derived from the muscle protein titin, even though cross-reactivity had not been predicted in preclinical studies (Linette GP et al. Blood 2013; 122:863-71, Cameron BJ, et al. Sci. Transl. Med. 2013; 5: 197-103). These patients demonstrated that TCR-engineered T cells may have severe, unpredictable off-target and organ-specific toxicity. [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] Therefore, there remains an unaddressed medical need to develop and provide antigen-binding proteins that bind specifically to targets with higher affinity, thus enabling targeting even tumor cells or cell lines with reduced expression of the target antigen peptide, while simultaneously maintaining a high safety profile due to low or reduced cross-reactivity with potential off-target peptides (also known as "simulating peptides" or "SimPeps"). Such antigen-binding proteins should also ideally have a good metabolic and / or pharmacokinetic profile and be suitable for large-scale production for industrial implementation. [Means for solving the problem]

[0008] Accordingly, the inventors designed an antigen-binding protein specific to the PRAME-004 peptide containing a CDR variant derived from the parent TCR R11P3D3. The antigen-binding protein provided herein exhibits increased binding affinity to the peptide-MHC complex, increased stability (e.g., reduced aggregation during expression and / or purification), and / or increased solubility, making it more suitable for medical applications.

[0009] Furthermore, the antigen-binding protein of the present invention (in particular, the bispecific T cell engaging receptor (TCER®)) exhibits high cytotoxicity against PRAME-004-positive tumor cells (e.g., cell lines Hs695T and U2OS cells), and reaches a maximum effective concentration of half dose (EC). 50 ) is 1-1000 pM. EC 50 This is 100 times higher, preferably more than 1000 times higher, compared to PRAME-004-negative tumor cells (e.g., cell line T98G), which indicates increased stability of the antigen-binding protein of the present invention.

[0010] Furthermore, the inventors demonstrated significant tumor growth inhibition at low doses in a therapeutic in vivo mouse model of the antigen-binding protein of the present invention.

[0011] In summary, our remarkable discovery provides the following advantages, particularly beyond the art: (i) increased affinity for target peptides while maintaining high tumor selectivity; (ii) increased specificity / reduced cross-reactivity resulting in reduced off-target and off-tumor cytotoxicity and an overall improvement in the safety profile; (iii) increased stability; (iv) improved expression yield and solubility suitable for large-scale production; and (v) the provision of an antigen-binding molecule having reduced immunogenicity.

[0012] In a first aspect, the present invention is an antigen-binding protein that specifically binds to a PRAME antigen peptide that contains or comprises the amino acid sequence SLLQHLIGL of SEQ ID NO: 50 and exists in complex with a major histocompatibility complex (MHC) protein, (a) Variable domain V including complementarity determination region (CDR) CDRa1, CDRa2, and CDRa3 A A first polypeptide comprising, The CDRa1 contains or comprises the amino acid sequence VKEFQD (Sequence ID 16), or an amino acid sequence different from Sequence ID 16 due to one, two, or three amino acid mutations, preferably amino acid substitutions. The CDRa3 contains an amino acid sequence different from sequence number 33 or sequence number 34, either by the amino acid sequence ALYNNLDMR (sequence number 33) or ALYNNYDMR (sequence number 34), or by one, two, or three (preferably one or two) amino acid mutations, preferably by amino acid substitutions. The first polypeptide and, (b) Variable domain V including CDRb1, CDRb2, and CDRb3 B A second polypeptide comprising, The aforementioned CDRb1 contains or comprises the amino acid sequence SGHNS (SEQ ID NO: 10), or one or two amino acid mutations, preferably amino acid substitutions, resulting in an amino acid sequence different from SEQ ID NO: 10. Said CDRb3 comprises or consists of the amino acid sequence ASSX1GX2X3DX4QY (SEQ ID NO: 327) (wherein X1 is P, A or T, X2 is A or S, X3 is T or I, and X4 is K or A), or an amino acid sequence that differs from SEQ ID NO: 327 by 1, 2 or 3 amino acid mutations, preferably amino acid substitutions, and a second polypeptide relates to an antigen-binding protein comprising

[0013] In a second aspect, the present invention relates to an isolated nucleic acid comprising a sequence encoding the antigen-binding protein of the first aspect of the present invention.

[0014] In a third aspect, the present invention relates to a vector comprising the nucleic acid of the second aspect of the present invention.

[0015] In a fourth aspect, the present invention relates to a host cell comprising the antigen-binding protein of the first aspect of the present invention, the nucleic acid of the second aspect, or the vector of the third aspect.

[0016] In a fifth aspect, the present invention relates to a pharmaceutical composition comprising the antigen-binding protein of the first aspect, the nucleic acid of the second aspect, the vector of the third aspect, or the host cell of the fourth aspect, and a pharmaceutically acceptable carrier.

[0017] In a sixth aspect, the present invention is a method for producing the antigen-binding protein of the first aspect of the present invention, comprising: (a) providing a host cell, (b) providing a gene construct comprising a coding sequence encoding the antigen-binding protein of the first aspect of the present invention, (c) introducing said gene construct into said host cell, and (d) expressing said gene construct by said host cell relates to a method comprising

[0018] In a seventh aspect, the present invention provides an antigen-binding protein of a first aspect, a nucleic acid of a second aspect, a vector of a third aspect, a host cell of a fourth aspect, or a pharmaceutical composition of a fifth aspect for pharmaceutical use (particularly for use in the diagnosis, prevention, and / or treatment of proliferative disorders). [Brief explanation of the drawing]

[0019] [Figure 1] Conversion of TCRs to stabilized scTCRs via yeast surface presentation. scTCR molecules presented on the surface of transformed Saccharomyces cerevisiae EBY100 were stained with anti-Myc-FITC antibody to determine expression levels, and functional binding was examined by staining with PE-labeled HLA-A*02 / PRAME-004 tetramer. The unmodified scTCR P11P3D3 (left panel, SEQ ID NO: 5) was compared to the R11P1D3_stabilized scTCR variant (right panel, SEQ ID NO: 6), obtained from a selection of scTCR libraries, which possesses nine stabilizing framework mutations and three single-point mutations in the CDR. [Figure 2] Affinity mutations in scTCR CDR1 alpha induced by yeast surface presentation. Stabilized scTCRs, including unmodified and mature CDR1 alpha, were stained with HLA-A*02 / PRAME-004 monomer at a concentration of 10 nM. Counter-staining was performed with a mixture of HLA-A*02 / SimPep tetramers containing peptides (sequence numbers 51-59) with high sequence similarity to PRAME-004 (sequence number 50) (each applied at a concentration of 10 nM). The stabilized scTCR R11P3D3SD (SEQ ID NO: 6), which has the unmodified alpha-chain CDR1 sequence SSNFYN (SEQ ID NO: 13; bottom right panel), is compared with scTCR variants containing affinity-mature alpha-chain CDR1 sequences VKEFQD, NKEFQD, TREFQD, NREFQD, TSEFQD, TKEFQD, VREFQD, TAEFQD, VSEFQD, VAEFQD, IKEFQN, VREFQN, and TAEFQN (SEQ ID NOs: 16-28), respectively. SSNFYN (SEQ ID NO: 13) is the corresponding CDRa1 sequence in the stabilized scTCR R11P3D3SD. [Figure 3] Binding of high-affinity scTCR yeast clones to similar peptides. Yeast clones possessing stable scTCRs with mature CDRs (SEQ ID NOs. 79-87 and 89-92) were stained with 100 nM HLA-A*02 monomers containing either the PRAME-004 target peptide or one of seven similar peptides (SEQ ID NOs. 52-56 and 58-59). [Figure 4] Binding of high-affinity scTCR yeast clones to similar peptides. Yeast clones possessing stable scTCRs with mature CDRs (SEQ ID NOs. 79-87 and 89-92) were stained with 100 nM HLA-A*02 monomers containing either the PRAME-004 target peptide or one of 19 similar peptides (SEQ ID NOs. 51, 57, 60, 62-69, and 71-78). R16P1C10_CDR6_scTCR (SEQ ID NOs. 357) was added as a reference, but for this clone, only binding to PRAME-004 and IFT17-003 (SEQ ID NOs. 60) was evaluated. [Figure 5-1] Determination of binding motifs using high-affinity scTCR yeast clones. Yeast clones possessing stable scTCRs with mature CDRs (SEQ ID NOs: 79, 80, 82, 83, and 85-87) were stained with PRAME-004 and PRAME-004 peptide variants containing alanine substitution (SEQ ID NOs: 318-324) under the application of HLA-A*02 at concentrations of 10 nM, 3 nM, 1 nM, and 0.3 nM. [Figure 5-2] Determination of binding motifs using high-affinity scTCR yeast clones. Yeast clones possessing stable scTCRs with mature CDRs (SEQ ID NOs: 79, 80, 82, 83, and 85-87) were stained with PRAME-004 and PRAME-004 peptide variants containing alanine substitution (SEQ ID NOs: 318-324) under the application of HLA-A*02 at concentrations of 10 nM, 3 nM, 1 nM, and 0.3 nM. [Figure 6-1]Screening of similar peptides to the soluble scTCR-Fab molecule. Binding to 14 similar peptides associated with HLA-A*02 (SEQ ID NOs: 187, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, and 212) was analyzed at a concentration of 1 μM scTCR-Fab using biolayer interferometry. The upper curve in each graph represents the binding of scTCR-Fab to the target HLA-A*02 / PRAME-004 monomer. [Figure 6-2] Screening of similar peptides to the soluble scTCR-Fab molecule. Binding to 14 similar peptides associated with HLA-A*02 (SEQ ID NOs: 187, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, and 212) was analyzed at a concentration of 1 μM scTCR-Fab using biolayer interferometry. The upper curve in each graph represents the binding of scTCR-Fab to the target HLA-A*02 / PRAME-004 monomer. [Figure 7] In vitro cytotoxicity of the TCER® molecule against target-positive and target-negative tumor cell lines. PBMCs derived from healthy HLA-A*02-positive donors were incubated with either the target-positive tumor cell line Hs695T (●) or the target-negative but HLA-A*02-positive tumor cell line T98G (○) at a 1:10 ratio, increasing the TCER® concentration. TCER®-induced cytotoxicity was quantified 48 hours after co-culture by measuring released LDH. The results of experiments evaluating TPP-93 and TPP-79 are shown in the lower and upper panels, respectively. [Figure 8]In vitro cytotoxicity of the TCER® molecule TPP-105 against target-positive and target-negative tumor cell lines. PBMCs derived from healthy HLA-A*02-positive donors were incubated with either the target-positive tumor cell line Hs695T (●) or the target-negative but HLA-A*02-positive tumor cell line T98G (○) at a 1:10 ratio, increasing the concentration of TPP-105. Cytotoxicity induced by TCER® was quantified 48 hours after co-culture by measuring released LDH. [Figure 9] Summary of cytotoxicity data for the TCER(registered trademark) Slot III molecule. EC50 values ​​of dose-response curves obtained from LDH release assays were calculated using nonlinear four-point curve fitting. For each evaluated TCER(registered trademark) molecule, the calculated EC50 values ​​are shown for target-positive tumor cell lines Hs695T (●), U20S (○), and target-negative but HLA-A*02-positive tumor cell line T98G (*). Therefore, each symbol represents a single assay utilizing PBMCs derived from various HLA-A*02-positive donors. For TPP-871 / T98G, the EC50 is an estimated value because T98G was not recognized by TPP-871. [Figure 10-1] In vitro cytotoxicity of the TCER(registered trademark) Slot III mutant against T2 cells loaded with various concentrations of target peptides. Cytotoxicity was determined by quantifying LDH released into the supernatant. Human PBMCs were used as effector cells in a 5:1 E:T ratio. Readouts were performed after 48 hours. [Figure 10-2] In vitro cytotoxicity of the TCER(registered trademark) Slot III mutant against T2 cells loaded with various concentrations of target peptides. Cytotoxicity was determined by quantifying LDH released into the supernatant. Human PBMCs were used as effector cells in a 5:1 E:T ratio. Readouts were performed after 48 hours. [Figure 10-3]In vitro cytotoxicity of the TCER(registered trademark) Slot III mutant against T2 cells loaded with various concentrations of target peptides. Cytotoxicity was determined by quantifying LDH released into the supernatant. Human PBMCs were used as effector cells in a 5:1 E:T ratio. Readouts were performed after 48 hours. [Figure 11-1] Safety analysis of normal tissue cells for selected TCER® Slot III variants. TCER®-mediated cytotoxicity against five different normal tissue cell types expressing HLA-A*02 was evaluated compared to cytotoxicity against PRAME-004-positive Hs695T tumor cells. PBMCs derived from healthy HLA-A*02+ donors were co-cultured in a 10:1 ratio with normal tissue cells or Hs695T tumor cells (triple) in a 1:1 mixture of normal tissue cell medium (4, 10a, or 13a) and T cell medium (LDH-AM), or co-cultured in T cell medium alone. After 48 hours, lysis of normal tissue cells and Hs695T cells was evaluated by measuring LDH release (LDH-Glo® Kit, Promega). [Figure 11-2] Safety analysis of normal tissue cells for selected TCER® Slot III variants. TCER®-mediated cytotoxicity against five different normal tissue cell types expressing HLA-A*02 was evaluated compared to cytotoxicity against PRAME-004-positive Hs695T tumor cells. PBMCs derived from healthy HLA-A*02+ donors were co-cultured in a 10:1 ratio with normal tissue cells or Hs695T tumor cells (triple) in a 1:1 mixture of normal tissue cell medium (4, 10a, or 13a) and T cell medium (LDH-AM), or co-cultured in T cell medium alone. After 48 hours, lysis of normal tissue cells and Hs695T cells was evaluated by measuring LDH release (LDH-Glo® Kit, Promega). [Figure 11-3]Safety analysis of normal tissue cells for selected TCER® Slot III variants. TCER®-mediated cytotoxicity against five different normal tissue cell types expressing HLA-A*02 was evaluated compared to cytotoxicity against PRAME-004-positive Hs695T tumor cells. PBMCs derived from healthy HLA-A*02+ donors were co-cultured in a 10:1 ratio with normal tissue cells or Hs695T tumor cells (triple) in a 1:1 mixture of normal tissue cell medium (4, 10a, or 13a) and T cell medium (LDH-AM), or co-cultured in T cell medium alone. After 48 hours, lysis of normal tissue cells and Hs695T cells was evaluated by measuring LDH release (LDH-Glo® Kit, Promega). [Figure 12-1] Safety analysis of normal tissue cells for selected TCER® Slot IV variants. TCER®-mediated cytotoxicity against 10 different normal tissue cell types expressing HLA-A*02 was evaluated compared to cytotoxicity against PRAME-004-positive Hs695T tumor cells. PBMCs derived from healthy HLA-A*02+ donors were co-cultured in a 10:1 ratio with normal tissue cells or Hs695T tumor cells (triple) in a 1:1 mixture of normal tissue cell medium (3, 4, 8a, 10a, 13a, or 16a) and T cell medium (LDH-AM), or co-cultured in T cell medium alone. After 48 hours, lysis of normal tissue cells and Hs695T cells was evaluated by measuring LDH release (LDH-Glo® Kit, Promega). [Figure 12-2]Safety analysis of normal tissue cells for selected TCER® Slot IV variants. TCER®-mediated cytotoxicity against 10 different normal tissue cell types expressing HLA-A*02 was evaluated compared to cytotoxicity against PRAME-004-positive Hs695T tumor cells. PBMCs derived from healthy HLA-A*02+ donors were co-cultured in a 10:1 ratio with normal tissue cells or Hs695T tumor cells (triple) in a 1:1 mixture of normal tissue cell medium (3, 4, 8a, 10a, 13a, or 16a) and T cell medium (LDH-AM), or co-cultured in T cell medium alone. After 48 hours, lysis of normal tissue cells and Hs695T cells was evaluated by measuring LDH release (LDH-Glo® Kit, Promega). [Figure 12-3] Safety analysis of normal tissue cells for selected TCER® Slot IV variants. TCER®-mediated cytotoxicity against 10 different normal tissue cell types expressing HLA-A*02 was evaluated compared to cytotoxicity against PRAME-004-positive Hs695T tumor cells. PBMCs derived from healthy HLA-A*02+ donors were co-cultured in a 10:1 ratio with normal tissue cells or Hs695T tumor cells (triple) in a 1:1 mixture of normal tissue cell medium (3, 4, 8a, 10a, 13a, or 16a) and T cell medium (LDH-AM), or co-cultured in T cell medium alone. After 48 hours, lysis of normal tissue cells and Hs695T cells was evaluated by measuring LDH release (LDH-Glo® Kit, Promega). [Figure 12-4]Analysis of normal tissue cell safety for selected TCER® Slot IV variants. TCER®-mediated cytotoxicity against 10 different normal tissue cell types expressing HLA-A*02 was evaluated in comparison with cytotoxicity against PRAME-004-positive Hs695T tumor cells. PBMCs derived from healthy HLA-A*02+ donors were co-cultured with normal tissue cells or Hs695T tumor cells (triplicate) at a ratio of 10:1 in a 1:1 mixture of respective normal tissue cell medium (3, 4, 8a, 10a, 13a, or 16a) and T cell medium (LDH-AM), or co-cultured in T cell medium alone. After 48 hours, lysis of normal tissue cells and Hs695T cells was evaluated by measuring LDH release (LDH-Glo™ Kit, Promega). [Figure 12-5] Analysis of normal tissue cell safety for selected TCER® Slot IV variants. TCER®-mediated cytotoxicity against 10 different normal tissue cell types expressing HLA-A*02 was evaluated in comparison with cytotoxicity against PRAME-004-positive Hs695T tumor cells. PBMCs derived from healthy HLA-A*02+ donors were co-cultured with normal tissue cells or Hs695T tumor cells (triplicate) at a ratio of 10:1 in a 1:1 mixture of respective normal tissue cell medium (3, 4, 8a, 10a, 13a, or 16a) and T cell medium (LDH-AM), or co-cultured in T cell medium alone. After 48 hours, lysis of normal tissue cells and Hs695T cells was evaluated by measuring LDH release (LDH-Glo™ Kit, Promega). [Figure 13-1]Safety analysis of selected TCER® Slot IV variants in normal tissue cells. TCER®-mediated cytotoxicity against six different normal tissue cell types expressing HLA-A*02 was evaluated compared to cytotoxicity against PRAME-004-positive Hs695T tumor cells. PBMCs derived from healthy HLA-A*02+ donors were co-cultured in a 10:1 ratio with normal tissue cells or Hs695T tumor cells (triple) in a 1:1 mixture of normal tissue cell medium (10a, 13a, or 16a) and T cell medium (LDH-AM). After 48 hours, lysis of normal tissue cells and Hs695T cells was evaluated by measuring LDH release (LDH-Glo® Kit, Promega). [Figure 13-2] Safety analysis of selected TCER® Slot IV variants in normal tissue cells. TCER®-mediated cytotoxicity against six different normal tissue cell types expressing HLA-A*02 was evaluated compared to cytotoxicity against PRAME-004-positive Hs695T tumor cells. PBMCs derived from healthy HLA-A*02+ donors were co-cultured in a 10:1 ratio with normal tissue cells or Hs695T tumor cells (triple) in a 1:1 mixture of normal tissue cell medium (10a, 13a, or 16a) and T cell medium (LDH-AM). After 48 hours, lysis of normal tissue cells and Hs695T cells was evaluated by measuring LDH release (LDH-Glo® Kit, Promega). [Modes for carrying out the invention]

[0020] definition "PRAME" or (an antigen preferentially expressed in melanoma) was initially identified as an antigen overexpressed in melanoma [Ikeda et al Immunity. 1997 Feb;6(2): 199-208]; it is also known as CT130, MAPE, and OIP-4 and has Uniprot accession number P78395 (available as of January 11, 2019). This protein functions as a receptor for retinoic acid receptor signaling [Epping et al., Cell. 2005 Sep 23; 122(6):835-47]. PRAME belongs to a family of germline-encoded antigens known as cancer-testis antigens. Cancer-testis antigens are attractive targets for immunotherapy interventions because their expression is typically limited or absent in normal adult tissues. PRAME is expressed in many solid tumors, as well as leukemia and lymphoma [Doolan et al., Breast Cancer Res Treat. 2008 May; 109(2):359-65;Epping et al., Cancer Res. 2006 Nov 15;66(22): 10639-42;Ercolak et al., Breast Cancer Res Treat. 2008 May; 109(2):359-65;Matsushita et al., Leuk Lymphoma. 2003 Mar;44(3):439-44;Mitsuhashi et al., Int. J Hematol. 2014; 100(1 ):88-95;Proto-Sequeire et al., Leuk Res. 2006 Nov;30(11): 1333-9;Szczepanski et al., Oral Oncol. 2013 Feb;49(2): 144-51;Van Baren et al., Br J Haematol. [1998 Sep; 102(5): 1376-9].The PRAME-targeted therapy of the present invention may be particularly suitable for the treatment of cancers including, but not limited to, acute myeloid leukemia, breast cancer, cholangiocarcinoma, gallbladder cancer, glioblastoma, hepatocellular carcinoma, head and neck squamous cell carcinoma, melanoma, achromatic melanoma, non-Hodgkin lymphoma, non-small cell lung cancer adenocarcinoma, non-small cell lung cancer, squamous cell non-small cell lung cancer, ovarian cancer, esophageal cancer, renal cell carcinoma, small cell lung cancer, bladder cancer, uterine cancer and endometrial cancer, chronic lymphocytic leukemia, colorectal cancer, osteosarcoma and synovial sarcoma, preferably breast cancer, cholangiocarcinoma, hepatocellular carcinoma, head and neck squamous cell carcinoma, squamous cell non-small cell lung cancer, ovarian cancer, esophageal cancer, renal cell carcinoma, small cell lung cancer, bladder cancer, uterine cancer and endometrial cancer, and synovial sarcoma.

[0021] The "PRAME antigen peptide" contains or consists of the amino acid sequence SLLQHLIGL (SEQ ID NO: 50), which corresponds to amino acids 425-433 of the full-length PRAME protein in the amino acid sequence of SEQ ID NO: 328, accessible under Uniprot accession number P78395 (available as of January 11, 2019). The PRAME-derived peptide containing or consisting of the amino acid sequence SLLQHLIGL (SEQ ID NO: 50) is also referred to herein as PRAME-004. The PRAME-004 peptide is a peptide epitope derived from tumor-associated or tumor-specific proteins and is presented on the cell surface by molecules of the major histocompatibility complex (MHC). More specifically, the PRAME-004-derived peptide is presented on the cell surface in complex with HLA-A*02. Med. 2001 Jan 1; 193(1):73-88. In relation to the present invention, the terms "PRAME antigen peptide," "PRAME peptide," or "PRAME-004" are used interchangeably and refer to a peptide containing or comprising the amino acid sequence SLLQHLIGL (SEQ ID NO: 50). Preferably, the PRAME peptide consists of the amino acid sequence SLLQHLIGL. If the PRAME peptide contains additional amino acids in addition to the amino acid sequence SLLQHLIGL, it is preferable that the total length of the PRAME peptide does not exceed 12 amino acids.

[0022] The terms “antigen” or “target antigen,” as used herein, refer to a molecule or portion of a molecule or complex to which an antigen-binding site can bind, the antigen-binding site being present, for example, in the antibodies, TCRs, and / or other antigen-binding proteins of the present invention. The antigens related to the present invention are PRAME peptides comprising or consisting of the amino acid sequence SLLQHLIGL of SEQ ID NO: 50, more specifically, PRAME peptides comprising or consisting of the amino acid sequence SLLQHLIGL of SEQ ID NO: 50 in a complex with an MHC protein (e.g., an HLA protein, e.g., HLA-A*02).

[0023] A "domain" can be any region of a protein, and is generally defined based on sequence homology, often associated with a specific structural or functional entity.

[0024] In relation to this invention, the term “immunoglobulin (Ig) domain” refers to a protein domain consisting of a two-layer sandwich of 7 to 9 antiparallel β-strands arranged in two β-sheets having a Greek key topology. The Ig domain is perhaps the most frequently used “building block” in naturally occurring proteins. Proteins containing Ig domains are incorporated into the immunoglobulin superfamily (e.g., antibodies, T cell receptors (TCRs), and cell adhesion molecules). Examples of Ig domains are the variable and constant domains of antibodies and TCRs.

[0025] V related to the present invention α This refers to the variable domain of the TCR α chain.

[0026] V related to the present invention β This refers to the variable domain of the TCR β chain.

[0027] V related to the present invention γ This refers to the variable domain of the TCR γ chain.

[0028] V related to the present inventionδ refers to the variable domain of a TCR δ chain.

[0029] V related to the present invention A refers to a variable domain comprising a TCR-derived CDR, specifically CDR1a, CDR3a derived from the α chain, and optionally CDR2a. The sequence surrounding the CDR (i.e., the framework sequence) may be derived from the variable domain of a TCR, that is, may be derived from the variable domain of a TCR α chain, β chain, γ chain, or δ chain, or may be derived from the variable domain of an antibody, and is preferably derived from the variable domain of a TCR α chain.

[0030] V related to the present invention B refers to a variable domain comprising a TCR-derived CDR, specifically CDR1b, CDR3b derived from the β chain, and optionally CDR2b. The sequence surrounding the CDR (i.e., the framework sequence) may be derived from the variable domain of a TCR, that is, may be derived from the variable domain of a TCR α chain, β chain, γ chain, or δ chain, or may be derived from the variable domain of an antibody, and is preferably derived from the variable domain of a TCR β chain.

[0031] V related to the present invention L refers to the variable domain of an antibody light chain.

[0032] V related to the present invention H refers to the variable domain of an antibody heavy chain.

[0033] C related to the present invention L refers to the constant domain of an antibody light chain.

[0034] C related to the present invention H1 , C H2 , and C H3 refer to the constant domains of an antibody heavy chain (specifically, an IgG heavy chain).

[0035] The term “epitope,” also known as an antigenic determinant, refers to the portion of an antigen recognized by the immune system. As used herein, the term epitope includes the terms “structural epitope” and “functional epitope.” A “structural epitope” is the amino acid of an antigen (e.g., a peptide-MHC complex) that, when bound to the antigen, is covered by the antigen-binding protein. Typically, all amino acids of the antigen that are within 5 Å of any amino acid of the antigen-binding protein are considered to be covered. The structural epitopes of an antigen can be determined by methods known in the art, including X-ray crystallography or NMR analysis. The structural epitopes of antibodies typically consist of 20–30 amino acids. The structural epitopes of TCRs typically consist of 20–30 amino acids. A "functional epitope" is a subset of amino acids that form a structural epitope and contains amino acids of the antigen that are important for forming an interface with the antigen-binding protein or its functional fragment of the present invention by directly forming non-covalent interactions (e.g., H-bonds, salt bridges, aromatic stacking, or hydrophobic interactions) or by indirectly stabilizing the antigen's binding conformation, and is determined, for example, by mutation scanning. In relation to the present invention, a functional epitope is also referred to as a "binding motif." Typically, the functional epitope of an antibody-bound antigen contains 4 to 6 amino acids. Typically, the functional epitope of a peptide-MHC complex contains 2 to 6 or 7 amino acids of the peptide and 2 to 7 amino acids of the MHC molecule. Since peptides presented by MHC I typically have 8 to 10 amino acids, only a subset of amino acids of each given peptide is part of the functional epitope of the peptide-MHC complex. The epitope (in particular, the functional epitope to which the antigen-binding protein of the present invention binds) contains or consists of amino acids of the antigen required for the formation of the binding interface. In relation to the present invention, the functional epitope (i.e., the binding motif) contains at least amino acids 3, 5, and 7 of the PRAME-004 antigen peptide of SEQ ID NO: 50, and preferably does not contain amino acids 1 and 4.

[0036] The major histocompatibility complex (MHC) is a set of cell surface proteins essential for the adaptive immune system to recognize foreign molecules in vertebrates, thereby determining histocompatibility. The primary function of MHC molecules is to bind to pathogen-derived antigens and present them on the cell surface for recognition by appropriate T cells. Human MHC is also called the HLA (human leukocyte antigen) complex (or simply HLA). The MHC gene family is divided into three subgroups: Class I, Class II, and Class III. Complexes of peptides with MHC Class I are recognized by CD8-positive T cells with the appropriate T cell receptor (TCR), while complexes of peptides with MHC Class II molecules are recognized by CD4-positive helper T cells with the appropriate TCR. Since both CD8-dependent and CD4-dependent responses work together synergistically to contribute to antitumor effects, the identification and characterization of tumor-associated antigens and their corresponding T cell receptors are crucial in the development of cancer immunotherapies such as vaccines and cell therapies. The HLA-A gene is located on a short arm of chromosome 6 and encodes the large α chain that is a component of HLA-A. Mutations in the HLA-A α chain are important for HLA function. These mutations promote genetic diversity in a population. Since each HLA has a different affinity for a particular peptide structure, a large number of HLA types means a large number of antigens that can be "presented" on the cell surface. The MHC class I HLA proteins related to this disclosure may be HLA-A proteins, HLA-B proteins, or HLA-C proteins, and are preferably HLA-A proteins, such as HLA-A*02. In MHC class I-dependent immune responses, peptides must not only be able to bind to a specific MHC class I molecule expressed by tumor cells, but also be subsequently recognized by T cells with a specific T cell receptor (TCR).

[0037] In this specification, "antigen peptide in a complex with an MHC protein" refers to an antigen peptide that is non-covalently bound to an MHC molecule. Specifically, the antigen peptide is located in a "peptide-binding groove" formed by the MHC molecule. The complex of an MHC molecule and an antigen peptide is also referred to herein as a "peptide-MHC complex" or "pMHC complex." In the case of the PRAME antigen peptide, this complex is also referred to as the "PRAME antigen peptide-MHC complex" or "PRME-004:MHC complex."

[0038] "HLA-A*02" indicates a specific HLA allele, the letter A indicates the allele, and the prefix "*02" indicates the A2 serotype.

[0039] In this specification, the term "antigen-binding protein" refers to a polypeptide containing an antigen-binding site capable of specifically binding to an antigen. The antigen-binding protein of the present invention comprises a TCR-derived CDR, specifically a variable domain V containing TCR-derived CDRa1, CDRa3, and optionally CDRa2. A and variable domain V, which includes TCR-derived CDRb1, CDRb3, and optionally CDRb2. B This includes V A Entire domain and / or V B The entire domain is derived from the TCR, therefore, V α Domain and V β Domain, or V γ Domain and V δ This is a domain. In the context of this specification, the term antigen-binding protein includes several TCR and antibody formats as defined below. In one example, the antigen-binding protein includes TCR-derived CDRs (specifically, TCR-derived CDRa1, CDRa3, CDRb1, CDRb3, and optionally CDRa2 and CDRb2) transplanted onto the heavy and light chains of the antibody. In another example, TCR-derived V α V from the entire domain and / or TCR βThe entire domain is transplanted into the heavy and light chains of the antibody. Those skilled in the art will recognize that such a construct represents a hybrid antigen-binding protein, which has the antigen specificity of the TCR from which the CDR or variable domain is derived, but has the overall structure of an antibody and can therefore be referred to as an “antibody.” The term antigen-binding protein further includes bispecific antigen-binding proteins or multispecific antigen-binding proteins. V includes CDRa1, CDRa3, CDRb1, CDRb3, and optionally CDRa2 and CDRb2, derived from the TCR as defined in the claims. A and V B In addition, such a bispecific antigen-binding protein or multispecific antigen-binding protein further comprises at least one variable domain and optionally a constant domain, the variable domain and / or constant domain may be derived from an antibody or TCR. Again, those skilled in the art will recognize that such a construct containing elements of both an antibody and a TCR represents a hybrid format and may be referred to as a “bispecific TCR,” a “bispecific antibody,” or a “bispecific TCR antibody molecule,” depending on the composition of the antigen-binding protein, but may also be referred to by the viewpoint and / or focus of those skilled in the art. In some embodiments, the antigen-binding protein of the present invention comprises CDRa1, CDRa3, CDRb1, CDRb3, and optionally CDRa2 and CDRb2 derived from the TCR as defined in the claims. A and V B including, V A or V BThe antigen-binding protein further includes additional domains directly or indirectly fused to it. Such antigen-binding proteins may be referred to as “TCR fusion proteins.” Examples of additional domains included in “TCR fusion proteins” are listed below. In preferred embodiments, the antigen-binding protein is a bispecific TCR-antibody molecule as defined below, and more preferably a bispecific T-cell engaging receptor [TCER®] as defined below. In such embodiments, the antigen-binding protein comprises two different antigen-binding sites and is capable of specifically binding to two different antigens simultaneously, for example, as known in bispecific antibodies.

[0040] In one embodiment, the antigen-binding protein of the present disclosure specifically binds to the PRAME antigen peptide, which contains or comprises the amino acid sequence SLLQHLIGL of SEQ ID NO: 50 and exists in complex with a major histocompatibility complex (MHC) protein, and this antigen-binding protein (a) Variable domain V including complementarity determination region (CDR) CDRa1, CDRa2, and CDRa3 A A first polypeptide comprising, CDRa1 contains an amino acid sequence VKEFQD (SEQ ID NO: 16), or an amino acid sequence different from SEQ ID NO: 16 by up to one, two, or three amino acid substitutions. CDRa3 contains an amino acid sequence different from sequence number 33 or sequence number 34 by the amino acid sequence ALYNNLDMR (sequence number 33) or ALYNNYDMR (sequence number 34), or by up to one, two, or three amino acid substitutions. CDRa2 contains an amino acid sequence FGPYGKE (SEQ ID NO: 32), or an amino acid sequence different from SEQ ID NO: 32 by up to one, up to two, or at least three amino acid substitutions. The first polypeptide and, (b) Variable domain V including CDRb1, CDRb2, and CDRb3 B A second polypeptide comprising, CDRb1 contains an amino acid sequence different from SEQ ID NO: 10, either the amino acid sequence SGHNS (SEQ ID NO: 10) or a sequence with up to one or up to two amino acid substitutions. CDRb3 contains an amino acid sequence different from sequence number 327 by the amino acid sequence ASSX1GX2X3DX4QY (sequence number 327) (wherein X1 is P, A, or T, X2 is A or S, X3 is T or I, and X4 is K or A), or by up to one, two, or three amino acid substitutions. CDRb2 contains the amino acid sequence FQNTAV (SEQ ID NO: 36), or a CDRb2 amino acid sequence that differs from SEQ ID NO: 36 by up to 1, 2, 3, 4, 5, or 6 amino acid substitutions. The second polypeptide and Includes.

[0041] In one embodiment, the antigen-binding protein of this disclosure is CDRa1 containing sequence number 16, CDRa2 containing sequence number 32, CDRa3 containing sequence number 33, CDRb1 containing sequence number 10, CDRb2 containing Sequence ID No. 36, and CDRb3 containing sequence number 327 Includes.

[0042] In one embodiment, the antigen-binding protein of this disclosure is CDRa1 containing sequence number 16, CDRa2 containing sequence number 32, CDRa3 containing sequence number 34, CDRb1 containing sequence number 10, CDRb2 containing Sequence ID No. 36, and CDRb3 containing Sequence ID 327 Includes.

[0043] In one embodiment, the amino acid substitution is a conservative amino acid substitution.

[0044] "At least one" in this specification means one or more designated subjects, for example, one, two, three, four, five, or six or more designated subjects. For example, "at least one binding site" in this specification means one, two, three, four, five, or six or more binding sites.

[0045] The term "bispecificity" in relation to the present invention refers to an antigen-binding protein having at least two valencies and binding specificities for two different antigens, and therefore containing at least two antigen-binding sites. The term "valency" refers to the number of binding sites of an antigen-binding protein; for example, a bivalent antigen-binding protein refers to an antigen-binding protein having two binding sites. The binding sites can bind to the same or different targets; that is, a bivalent antigen-binding protein may be monospecific (i.e., bind to one target) or bispecific (i.e., bind to two different targets). The antigen-binding molecule of the present invention contains at least one antigen-binding site, including a TCR-derived CDR. In a preferred embodiment, the antigen-binding molecule of the present invention contains at least one TCR-derived antigen-binding site.

[0046] As used herein, the term "TCR" means conventional / native TCRs and engineered TCRs, in particular functional TCR fragments, single-stranded TCRs, and bispecific or multispecific TCRs.

[0047] "Native TCR" refers to wild-type TCR that can be isolated from nature. TCRs that have the same domains and domain configuration as native TCRs and that include TCR-derived CDRs and framework regions may also be called "conventional TCRs." Native / conventional TCRs are heterodimeric cell surface proteins of the immunoglobulin superfamily, which associate with an invariant protein of the CD3 complex involved in signal transduction. Native heterodimeric TCRs exist in αβ and γδ forms, which are structurally similar but differ in location and possibly function. The extracellular portions of native heterodimeric αβTCRs and γδTCRs contain two polypeptides, each having a membrane-proximal constant domain (also called a constant region) and a membrane-distal variable domain (also called a variable region). In relation to the present invention, such TCRs are also called full-length TCRs. Native αβ heterodimeric TCRs have an α chain and a β chain. The α-chain comprises a variable (V) region, a binding (J) region, and a constant (C) region, while the β-chain comprises the V, J, and C regions, and usually further includes a short diversity (D) region between the variable and binding regions, although this diversity region is often considered part of the binding region. The constant regions of the TCR α-chain and β-chain are referred to as TRAC and TRBC, respectively (Lefranc, (2001), Curr Protoc Immunol Appendix 1: Appendix 10). In relation to the present invention, the constant regions of the TCR α-chain and β-chain (TRAC and TRBC) include a transmembrane (TM) region. Each of the constant and variable regions (or domains) contains an intrachain disulfide bond. The variable domain contains a highly polymorphic loop similar to the complementarity-determining region (CDR) of an antibody.

[0048] Each TCR variable domain contains three “TCR complementarity-determining regions” (CDRs) embedded in the framework sequence, one of which is a hypervariable region named CDR3. In relation to the present invention, CDRa1, CDRa2, and CDRa3 represent α-chain CDRs, and CDRb1, CDRb2, and CDRb3 represent β-chain CDRs. Several types of α-chain and β-chain variable domains exist, distinguished by the framework, CDR1, and CDR2 sequences, as well as the partially defined CDR3 sequence. Alpha-chain variable domain types are referred to by unique TRAV numbers in IMGT nomenclature, and beta-chain variable domain types are referred to by unique TRBV numbers in IMGT nomenclature (Folch and Lefranc, (2000), Exp Clin Immunogenet 17(1): 42-54; Scaviner and Lefranc, (2000), Exp Clin Immunogenet 17(2): 83-96; LeFranc and LeFranc, (2001), "T cell Receptor Factsbook", Academic Press). Further information on antibodies and TCR genes can be found in the international ImMunoGeneTics information system (registered trademark), Lefranc MP et al., (Nucleic Acids Res. 2015 Jan;43(Database issue):D413-22; and http: / / www.imgt.org / ). In the TCR, the CDR1 and CDR3 amino acid residues contact the antigen peptide, while the CDR2 amino acid residue primarily contacts the HLA molecule (Stadinski et al., J Immunol. 2014 June 15; 192(12): 6071-6082; Cole et al., J Biol Chem. 2014 Jan 10;289(2):628-38). Therefore, the antigen specificity of the TCR is defined by the CDR3 and CDR1 sequences. The CDR2 sequence is not necessary for determining antigen specificity, but it may play a role in the overall affinity of the TCR to the peptide:MHC complex.

[0049] The "TCR framework region" (FR) refers to the amino acid sequence sandwiched between CDRs, i.e., the portion of the variable domain that is conserved to some extent between different TCRs. The α-chain, β-chain, γ-chain, and δ-chain variable domains each have four FRs, which are referred to herein as FR1-a, FR2-a, FR3-a, FR4-a (for the α-chain or γ-chain) and FR1-b, FR2-b, FR3-b, FR4-b (for the β-chain or δ-chain), respectively. Thus, the α-chain or γ-chain variable domain may be described as (FR1-a)-(CDRa1)-(FR2-a)-(CDRa2)-(FR3-a)-(CDRa3)-(FR4-a), and the β-chain or δ-chain variable domain may be described as (FR1-b)-(CDRb1)-(FR2-b)-(CDRb2)-(FR3-b)-(CDRb3)-(FR4-b). In relation to the present invention, the CDR / FR sequence in the α, β, γ, or δ chain variable domain is determined based on the IMGT definition (Lefranc et al., Dev. Comp. Immunol., 2003, 27(1):55-77; www.imgt.org). Accordingly, the CDR / FR amino acid position, when related to the TCR or a TCR-derived domain, is indicated according to the IMGT definition. Preferably, the variable domain V α The IMGT positions of the CDR / FR amino acid positions are assigned similarly to the IMGT numbering of TRAV24*01, and / or variable domain V β The IMGT positions of the CDR / FR amino acid positions are assigned in a manner similar to the IMGT numbering of TRBV12-3*01.

[0050] In relation to the present invention, the term "α / β TCR / CD3 complex" refers to a T cell receptor complex such as those present on the surface of T cells. Most T cells express an α / β TCR composed of disulfide-bonded α and β chains, which typically binds to the complex surface of an antigen peptide presented by MHC. The TCR constitutively associates with CD3, a protein complex that does not generate signaling on its own but is called a T cell coreceptor and contains an intracellular signaling motif (Birnbaum et al.; PNAS vol. 11, no. 49; 17576-17581, 2014). The α / β TCR is non-covalently coupled to this conserved multi-subunit signaling apparatus, which includes CD3εγ, CD3εδ, and CD3ζζ dimers (together forming the α / β TCR / CD3 complex).

[0051] "CD3" is a protein complex composed of four distinct chains. In mammals, this complex includes the CD3γ chain, CD3δ chain, and two CD3ε chains. These chains associate with the TCR and ζ chain, generating activation signals in T lymphocytes.

[0052] An engineered TCR (and therefore, as used in connection with the present invention, the term "TCR") includes, in particular, functional TCR fragments, stable mature TCRs, affinity mature TCRs, single-stranded TCRs, chimeric, humanized, bispecific, and multispecific TCRs. A "functional TCR fragment" includes (a) a fragment of a native or conventional TCR that retains the ability derived from the TCR to bind to a target antigen, and (b) a recombinant / engineered antigen-binding protein containing TCR-derived CDR sequences, in particular CDR1, CDR3, and optionally CDR2 sequences. Since binding to the target antigen is defined by these CDR sequences, an antigen-binding protein containing these CDR sequences retains the ability of the TCR from which the CDRs originate to bind to the target antigen. Those skilled in the art will recognize that while the CDR must contain scattered framework regions (FRs), their specific amino acid sequences are not important for the specificity of the target antigen. Therefore, a variable domain containing TCR-derived CDRs and antibody-derived FRs may be considered a "functional TCR fragment." Further examples of functional TCR fragments include single variable domains, e.g., V α , V β , V δ , V γ , or fragments of α, β, δ, γ chains, for example, "V α -C α " or "V β -C β Examples include, or parts thereof. Such fragments may also further include a corresponding hinge region. "Single-stranded TCR (scTCR)" as used herein refers to a TCR in which the variable domain of the TCR is located on a single polypeptide. Typically, the variable domain in an scTCR is separated by a linker, which typically contains 10 to 30 amino acids, for example, 25 amino acids.

[0053] In this specification, "chimeric TCR" refers to a TCR whose TCR chain contains sequences derived from multiple species. Preferably, the TCRs related to the present invention may include an α chain containing the human variable region of the α chain (e.g., the mouse constant region of the mouse TCR α chain). "Bispecific TCR" refers to a bispecific TCR-antibody molecule, particularly scTCR-Fab , or including a T cell engaging receptor [TCER®] as defined below.

[0054] As used herein, the term "antibody" means conventional / native antibodies and engineered antibodies, in particular functional antibody fragments, single-chain antibodies, single-domain antibodies, bispecific or multispecific antibodies.

[0055] "Native antibodies" refer to wild-type antibodies that can be isolated from nature. Antibodies that have the same type of domains and domain configuration as native antibodies and also contain antibody-derived CDR and FR sequences may also be called "conventional antibodies." In native / conventional antibodies, two heavy chains are linked to each other by disulfide bonds, and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chains: lambda (λ) and kappa (κ). There are five main heavy chain classes (or isotypes) that determine the functional activity of antibody molecules: IgM, IgD, IgG, IgA, and IGE. Each chain contains different domains (also called regions). The light chain has a variable domain (V L ) and constant domain (C L It contains two domains, ) and . The heavy chain contains the following four or five domains depending on the antibody isotype: variable domain (V H ), as well as 3 or 4 constant domains (collectively C H C H1 , C H2 , and C H3 , C as appropriate H4 ). Light (V L ) Chain and weight (V H Both variable domains of the chain determine binding recognition and specificity to the antigen. L ) Chain and heavy (C H The constant domain of the chain is involved in the association, secretion, placental transfer, complement binding, and Fc receptor (F c It confers important biological properties, such as binding to R).

[0056] Antibody specificity lies in the structural complementarity between the antibody binding site and the antigenic determinant. The antibody binding site is primarily composed of residues derived from the "antibody complementarity-determining region" (CDR) or hypervariable region. Occasionally, residues from the non-hypervariable region or framework region (FR) affect the overall domain structure and, consequently, the binding site. CDR refers to the amino acid sequence that defines the binding affinity and specificity of the native Fv region of the native antibody binding site. The light and heavy chains of an antibody each have three CDRs, called CDR1-L, CDR2-L, CDR3-L, and CDR1-H, CDR2-H, and CDR3-H, respectively. Therefore, the antigen-binding site of an antibody contains six CDRs, including the CDR sets derived from the heavy chain V region and the light chain V region, respectively. The "antibody framework region" (FR) refers to the amino acid sequence interposed between CDRs, i.e., the relatively conserved portion of the variable regions of the antibody light and heavy chains across different antibodies within a single species. The light and heavy chains of the antibody each have four FRs, called FR1-L, FR2-L, FR3-L, FR4-L, and FR1-H, FR2-H, FR3-H, and FR4-H, respectively. Thus, the light chain variable domain can be described as (FR1-L)-(CDR1-L)-(FR2-L)-(CDR2-L)-(FR3-L)-(CDR3-L)-(FR4-L), and the heavy chain variable domain can be described as (FR1-H)-(CDR1-H)-(FR2-H)-(CDR2-H)-(FR3-H)-(CDR3-H)-(FR4-H). As used herein, "human framework region" means a framework region that is substantially identical (about 85% or more, in particular 90%, 95%, 97%, 99%, or 100%) to the framework region of a naturally occurring human antibody. In relation to the present invention, the CDR / FR definitions in the variable domain of an antibody light or heavy chain are determined based on the IMGT definition (Lefranc et al., Dev. Comp. Immunol., 2003, 27(1):55-77; www.imgt.org). Accordingly, the amino acid sequences of CDR1, CDR2, and CDR3, as well as the amino acid sequences of FR1, FR2, FR3, and FR4 of a given variable chain are shown according to the IMGT definition.

[0057] The manipulated antibody formats include functional antibody fragments, single-chain antibodies, single-domain antibodies, and chimeric antibodies, humanized antibodies, bispecific antibodies, or multispecific antibodies. The manipulated antibody formats further include constructs in which a TCR-derived CDR (possibly containing an additional 3, 2, or 1 N and / or C-terminal framework residue) or an entire TCR-derived variable domain is transplanted onto the antibody heavy or light chain. More specifically, CDRa1, CDRa3, and optionally CDRa2 may be transplanted into the variable heavy chain amino acid sequence, and CDRb1, CDRb3, and optionally CDRb2 may be transplanted into the variable light chain amino acid sequence, and vice versa. As another example, the light chain variable domain of an antibody may be replaced with the α-chain variable domain of the TCR, and the heavy chain variable domain may be replaced with the β-chain variable domain of the TCR, and vice versa. "Functional antibody fragment" refers to the portion of a full-length antibody that retains the ability to bind to a target antigen, in particular the antigen-binding region or variable region of a full-length antibody. Examples of functional antibody fragments include Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2, and diabodies. Functional antibody fragments can also be single-domain antibodies such as heavy-chain antibodies. The term "Fab" refers to an antibody fragment obtained by treating IgG with a protease (e.g., papain), in which approximately half of the N-terminal side of the H chain and the entire L chain are linked to each other via disulfide bonds, with a molecular weight of approximately 50,000 daltons and antigen-binding activity. The Fv fragment is the N-terminal portion of the Fab fragment of an antibody, consisting of a variable portion of one light chain and one heavy chain.

[0058] As used herein, the “format” of an antigen-binding protein specifies a defined spatial arrangement of domains (in particular, variable domains and, as appropriate, constant domains). Key features of such an antigen-binding protein format are: the number of polypeptide chains (single-stranded, double-stranded, or multi-stranded), the type and length of the linkers connecting the different domains, the number of variable domains (and therefore the number of valencies), the number of different variable domains (and therefore the number of specificities for different antigens, e.g., bispecificity, multispecificity), and the order and orientation of the variable domains (e.g., crossover, parallel).

[0059] Many different bispecific and multispecific formats have been described in the art in relation to antibodies, and as will be understood by those skilled in the art, such bispecific and multispecific formats can be used in relation to the present invention by replacing the antibody domains of these formats with the variable domains described in relation to the present invention. Examples of such formats include diabodies, crossover bivariable domains (CODVs), and bivariable domain (DV) proteins. An overview of various bispecific antibody formats and methods for producing them is disclosed, for example, in Brinkmann U. and Kontermann EE MAbs. 2017 Feb-Mar; 9(2): 182-212. The DVD format is disclosed in the following scientific publications, for example: (Wu C et al. Nat Biotechnol 2007; 25:1290-7; PMID:17934452; Wu C. et al. MAbs 2009; 1:339-47; Lacy SE et al. MAbs 2015; 7:605-19; PMID:25764208; Craig RB et al. PLoS One 2012; 7:e46778; PMID:23056448; Piccione EC et al. MAbs 2015). CODV is disclosed, for example, in Onuoha SC et al. Arthritis Rheumatol. 2015 Oct; 67(10):2661-72 or, for example, in International Publication No. 2012 / 135345 and International Publication No. 2016 / 116626.Diabody is, for example, Holliger P et al. Protein Eng 1996; 9:299-305; PMID:8736497; Atwell JL et al. Mol Immunol 1996; 33:1301-12; PMID:9171890; Kontermann RE, Nat Biotechnol 1997; 15:629-31; PMID:9219263; Kontermann RE et al. Immunotechnology 1997; 3:137-44; PMID:9237098; Cochlovius B et al. Cancer Res 2000; 60:4336-41; PMID:10969772; and DeNardo DG et al. Cancer Biother Radiopharm 2001; 16:525-35; This is explained in PMID:11789029.

[0060] A "diabody" refers to a bivalent molecule consisting of two chains, each containing two variable domains, either derived from the same antibody or from different antibodies. When the antibodies are different, typically one antibody (V) is used. LX and V HX The variable domain of antibody X) is located on two different polypeptide chains, and the other antibody (V) LY and V HY The variable domain of antibody Y, which contains [substance name], is also located on two different polypeptide chains. These domains dimerize in a head-to-tail direction. The two chains can have the following structure: V HX -L Db1 -V LY and V HY -L Db2 -V LX , or V LX -L Db1 -V HY and V LY -L Db2 -V HX , or V HX -L Db1 -V HY and V LY -L Db2 -VLX , or V LX -L Db1 -V LY and V HY -L Db2 -V HX . To allow head-to-tail dimerization of domains, the two chains comprise linkers that separate the variable domains and may be the same or different (i.e., L Db1 and L Db2 ). This linker is preferably a short linker. Short linkers are typically 2 to 12, 3 to 13, for example 3, 4, 5, 6, 7, 8, 9 amino acids in length, for example 4 or 5 [Brinkmann U. and Kontermann E.E. (MAbs. 2017 Feb-Mar; 9(2): 182-212], or 8 amino acids in length, for example "GGGS" of SEQ ID NO: 290, "GGGGS" of SEQ ID NO: 286, or "GGGSGGGG" of SEQ ID NO: 214.

[0061] In the "dual variable domain immunoglobulin" [DVD-Ig™] format, the target-binding variable domains (domains V LY and V HY ) are typically fused to a conventional antibody X (comprising domains V LX and V HX ), such that the light chain of this conventional antibody X comprises an additional light chain variable domain (V LY ), and the heavy chain of conventional antibody X comprises an additional heavy chain variable domain (V HY ). The DVD-Ig™ described in the art typically comprises the following two polypeptide chains: one heavy chain comprising V HY -L1-V HX -L2-C H1 -C H2 -C H3 , and one light chain comprising V LY -L3-V LX -L4-C L , or V HX -L1-V HY -L2-C H1 -C H2 -CH3 A single heavy chain containing V LX -L3-V LY -L4-C L It is composed of a single light chain containing [a specific component]. Therefore, Domain V HY / V LY and V HX / V LX These form parallel pairs. Linked linkers L1 and L3 are preferably 5 to 20 amino acids, for example 5 to 15 amino acids, and / or linked linkers L2 and L4 may or may not be present.

[0062] The “crossover dual variable domain” (CODV) format described in the relevant technical field is a variable domain (V) of antibody X. LX and V HX ) and the variable domain of antibody Y (V LY and V HY ) represents a format in which these variable domains are concatenated in a way that enables crossover pair formation.

[0063] In the CODV-Ig format related to the present invention, the polypeptide chain has, for example, the following structure: V HX -L1-V HY -L2-C H1 -C H2 -C H3 and V LY -L3-V LX -L4-C L , or V HY -L1-V HX -L2-C H1 -C H2 -C H3 and V LX -L3-V LY -L4-C L , or V HX -L3-V HY -L4-C H1 -C H2 -C H3 and V LY -L1-V LX -L2-C L , or VHY -L3-V HX -L4-C H1 -C H2 -C H3 and V LX -L1-V LY -L2-C L The linked linkers (L1-L4) (also called total glycine linkers or serine-glycine linkers) typically have different lengths. To enable crossover pair formation, one chain (heavy or light chain) typically contains linkers that are longer than the other chains. For example, in the CODV structures listed above, L1 is the length of 3-12 amino acid residues, L2 is the length of 3-14 amino acid residues, L3 is the length of 1-8 amino acid residues, and L4 is the length of 1-3 amino acid residues, or L1 is the length of 5-10 amino acid residues, L2 is the length of 5-8 amino acid residues, L3 is the length of 1-5 amino acid residues, and L4 is the length of 1-2 amino acid residues, or L1 is the length of 7 amino acid residues, L2 is the length of 5 amino acid residues, L3 is the length of 1 amino acid residue, and L4 is the length of 2 amino acid residues.

[0064] The term "humanized antibody" refers to an antibody that is entirely or partially of non-human origin and has been modified by replacing certain amino acids (particularly in the heavy and light chain framework regions) to avoid or minimize the immune response in humans. The constant domain of a humanized antibody is primarily human C11. H Domain and C L This is a domain. Numerous methods for humanizing antibody sequences are known in the art; see, for example, the overview by Almagro & Fransson (2008) Front Biosci. 13: 1619-1633.

[0065] As will be understood by those skilled in the art, the structure of the antibody, in particular the structure of the heavy chain variable domain and light chain variable domain of the antibody, is similar to the structure of the variable domains of the TCR α, β, γ, or δ chain, which facilitates the transplantation of CDRs into antibodies (e.g., conventional antibodies, bispecific antibodies, or multispecific antibodies) as defined in relation to the present invention.

[0066] By knowing the amino acid sequence of the CDR of the antibody, TCR, or antigen-binding protein of the present invention, a person skilled in the art can easily determine the framework region (e.g., the TCR framework region or the antibody framework region). If the CDR is not shown, a person skilled in the art can first determine the CDR amino acid sequence based on the IMGT definition for the TCR or the IMGT definition for the antibody, and then determine the amino acid sequence of the framework region.

[0067] Bispecific TCR-antibody format In preferred embodiments, the antigen-binding protein of the present invention is a bispecific molecule, and more particularly, a bispecific TCR-antibody molecule, i.e., an antigen-binding protein comprising at least two antigen-binding sites, one of which is derived from an antibody and the other from or derived from a TCR or a TCR-derived CDR (particularly CDR1a, CDR3a, CDR1b, CDR3b, and optionally CDR2a and CDR2b). The antigen-binding site derived from the antibody has a variable domain V L and V H Includes.

[0068] In such bispecific TCR-antibody molecules, the variable domain may be arranged as described, for example, with respect to the various bispecific antibody formats described above. Techniques for producing such bispecific antibodies are also disclosed in the prior art cited above, and therefore, those skilled in the art can easily generate and produce the antigen-binding proteins of the present invention in the formats disclosed herein using the CDR or variable domain as defined herein. In addition, further formats are possible, for example, in which on each chain the variable domain is separated by a constant domain that mediates dimerization, and as a result, in the final molecule, two antigen-binding sites are located on either side of the dimerizing constant domain. Those skilled in the art can fully select an appropriate linker to ensure that the molecules fold reliably into the desired three-dimensional structure.

[0069] In its most preferred embodiment, the antigen-binding protein of the present invention is a bispecific T-cell engaging receptor [TCER®], which is a soluble Fc-containing bispecific antigen-binding molecule containing a TCR antigen-binding site and an antibody-antigen-binding site. The antibody-antigen-binding site is formed by the heavy chain variable region and light chain variable region of the antibody and is also called a "recruiter" because it binds to effector cells (e.g., T cells) and recruits them to tumors. TCER® comprises two polypeptide chains, and the antigen-binding site is formed by variable domains located on different polypeptide chains in a crossover orientation.

[0070] In the context of this application, a sequence that is "at least 85% identical to the reference sequence" means a sequence that has 85% or more, and in particular 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the full length of the reference sequence. A protein consisting of an amino acid sequence that is "at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical" to the reference sequence may contain mutations (e.g., deletions, insertions, and / or substitutions) compared to the reference sequence. In the case of substitutions, a protein consisting of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the reference sequence may correspond to a homologous sequence originating from a different species than the reference sequence.

[0071] In the context of this application, the “identity ratio” can be calculated using global pairwise alignment (i.e., two sequences are compared over their entire length). Methods for comparing the identity of two or more sequences are known in the art. For example, one could use the Needleman-Wunsch global alignment algorithm (Needleman and Wunsch, 1970 J. Mol. Biol. 48:443-453) to find the optimal alignment (including gaps) of two sequences considering their entire length. The Needle program is available, for example, on the ebi.ac.uk World Wide Web site and is further described in the following publication: [EMBOSS: The European Molecular Biology Open Software Suite (2000) Rice, P. Longden, I. and Bleasby, A. Trends in Genetics 16, (6) pp. 276-277]. The degree of identity between two polypeptides according to the present invention is calculated using the EMBOSS:Needle(Global) program with a “Gap Open” parameter of 10.0, a “Gap Extend” parameter of 0.5, and a Blosum62 matrix.

[0072] An amino acid substitution may be conservative or non-conservative. Preferably, the substitution is a conservative substitution in which one amino acid is replaced by another amino acid that has similar structure and / or chemical properties.

[0073] In one embodiment, a conservative substitution might be one described by Dayhoff in "The Atlas of Protein Sequence and Structure. Vol. 5", Natl. Biomedical Research (this content is incorporated in its entirety by reference). For example, in a certain embodiment, amino acids belonging to one of the following groups can be exchanged with each other, thus constituting a conservative exchange: Group 1: alanine (A), proline (P), glycine (G), asparagine (N), serine (S), threonine (T); Group 2: cysteine ​​(C), serine (S), tyrosine (Y), threonine (T); Group 3: valine (V), isoleucine (I), leucine (L), methionine (M), alanine (A), phenylalanine (F); Group 4: lysine (K), arginine (R), histidine (H); Group 5: phenylalanine (F), tyrosine (Y), tryptophan (W), histidine (H); and Group 6: aspartic acid (D), glutamic acid (E). In some embodiments, the conservative amino acid substitution may be selected from T→A, G→A, A→I, T→V, A→M, T→I, A→V, T→G, and / or T→S.

[0074] In further embodiments, conservative amino acid substitutions may include substitutions of an amino acid with another amino acid of the same class [e.g., (1) nonpolar: Ala, Val, Leu, Ile, Pro, Met, Phe, Trp; (2) non-charged: Gly, Ser, Thr, Cys, Tyr, Asn, Gln; (3) acidic: Asp, Glu; and (4) basic: Lys, Arg, His]. Other conservative amino acid substitutions may also be made as follows: (1) aromatic: Phe, Tyr, His; (2) proton donors: Asn, Gln, Lys, Arg, His, Trp; and (3) proton acceptors: Glu, Asp, Thr, Ser, Tyr, Asn, Gln (see, for example, U.S. Patent No. 10,106,805, which is incorporated by reference in its entirety).

[0075] In another embodiment, conservative substitutions may be performed according to Table 1. A method for predicting resistance to protein modification can be found, for example, in Guo et al., Proc. Natl. Acad. Sci., USA, 101(25):9205-9210 (2004) (this content is incorporated in its entirety by reference).

[0076] [Table 1]

[0077] In another embodiment, the conservative substitutions may be those shown in Table 2 under the heading "Conservative Substitutions." If such substitutions alter biological activity, more substantial changes, indicated as "Exemplary Substitutions" in Table 2, may be introduced, and the products may be screened as needed.

[0078] [Table 2]

[0079] The antigen-binding protein of the present invention may be of any length and may contain any number of amino acids, as long as it retains biological activity (e.g., the ability to specifically bind to a target antigen, the ability to detect diseased cells in a host, or the ability to treat or prevent a host disease).

[0080] The antigen-binding proteins of the present invention may contain synthetic amino acids instead of one or more naturally occurring amino acids. Such synthetic amino acids are known in the art and include, for example: aminocyclohexanecarboxylic acid, norleucine, α-amino n-decanoic acid, homoserine, S-acetylaminomethylcysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyllysine, N',N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptane Carboxylic acids, α-(2-amino-2-norbornane)-carboxylic acid, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine, and α-tert-butylglycine.

[0081] In one embodiment, the antigen-binding protein of the present invention may be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized, for example via disulfide crosslinks, converted to an acid addition salt, and / or dimerized, polymerized, or conjugated.

[0082] In further embodiments, the antigen-binding protein of the present invention is in the form of a salt, for example, a pharmaceutically acceptable salt. Suitable pharmaceutically acceptable acid addition salts include those derived from mineral acids (e.g., hydrochloric acid, hydrobromic acid, phosphoric acid, metaphosphoric acid, nitric acid, and sulfuric acid), as well as those derived from organic acids (e.g., tartaric acid, acetic acid, citric acid, malic acid, lactic acid, fumaric acid, benzoic acid, glycolic acid, gluconic acid, succinic acid, and arylsulfonic acids, such as p-toluenesulfonic acid).

[0083] In this specification, "covalent linkage" refers to peptide linkage or covalent linkage via a linker or linker sequence, such as a disulfide crosslink or a polypeptide linker.

[0084] As used herein, the term "linker" refers to one or more amino acid residues inserted between domains or between a domain and a drug to provide sufficient mobility for a domain or element (e.g., the variable domain of the antigen-binding protein of the present invention) to fold correctly to form an antigen-binding site in, for example, a crossover pair formation of an antigen-binding protein (in some of the CODV format or Diabody format) or a parallel pair formation configuration (e.g., in the DVD format).

[0085] In some embodiments, the linker consists of zero amino acids, meaning that no linker is present. Linkers are inserted at the amino acid sequence level in transfers between variable domains or between variable domains and constant domains (or between dimerized domains). Since the approximate sizes of the antibody domain and TCR domain are well understood, transfers between domains can be identified. The precise location of domain transfers can be determined by positioning peptide stretches that do not form secondary structure elements such as β-sheets or α-helices, as demonstrated by experimental data or as can be assumed by modeling or secondary structure prediction techniques. The term linker as used in relation to the present invention includes, but is not limited to, linkers referred to as L1, L2, L3, L4, L5, and L6.

[0086] Linkers such as L1, L2, L3, L4, L5, and L6 may have a length of at least 1 to 30 amino acids unless otherwise specified in their respective contexts. In some embodiments, linkers such as L1, L2, L3, L4, L5, and L6 may have a length of 2 to 25, 2 to 20, or 3 to 18 amino acids. In some embodiments, linkers such as L1, L2, L3, L4, L5, and L6 may be peptides with a length of 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 or fewer amino acids. In other embodiments, linkers such as L1, L2, L3, L4, L5, and L6 may have a length of 5 to 25, 5 to 15, 4 to 11, 10 to 20, or 20 to 30 amino acids. In other embodiments, linkers such as L1, L2, L3, L4, L5, and L6 may have lengths of approximately 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, or 30 amino acids. In specific embodiments, linkers such as L1, L2, L3, L4, L5, and L6 may have lengths of less than 24, less than 20, less than 16 amino acid residues, less than 12, less than 10, for example, 5 to less than 24, 10 to less than 24, or 5 to less than 10 amino acid residues. In some embodiments, the linker is the length of one or more amino acid residues, for example, the length of more than one, more than two, more than five, more than ten, more than twenty amino acid residues, and more than twenty twenty amino acid residues.

[0087] Exemplary linkers such as L1, L2, L3, L4, L5, and L6 contain or consist of amino acid sequences selected from the group consisting of GGGS (SEQ ID NO: 290), GGGGS (SEQ ID NO: 286), GGGAS (SEQ ID NO: 287), GGGSGGGG (SEQ ID NO: 214), GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 61), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 70), GGSGG (SEQ ID NO: 226), GGGGSGGGGSGGGGS (SEQ ID NO: 280), GGGGSAAA (SEQ ID NO: 358), and in particular GGGSGGGG (SEQ ID NO: 214), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 70), and GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 61).

[0088] "F c The term "domain" is used in connection with the present invention, and is a native F c and F c It includes mutants, monomeric dimers and polymeric F, whether digested from the whole antibody or generated by other means. c Includes both domains.

[0089] "Native F" c The term "antigen-binding fragment," as used herein, refers to a molecule containing a sequence of non-antigen-binding fragments, whether in monomeric, dimeric, or polymeric form, resulting from or generated by the digestion of an antibody, and which may include a hinge region. c The original antibody source is of human origin and can be any antibody class, but IgG1 and IgG2 are preferred. Native F c The molecule is composed of monomeric polypeptides that can be linked into dimeric or polymeric forms through covalent (i.e., disulfide bond) and non-covalent associations. Native F cThe number of intermolecular disulfide bonds between monomeric subunits of a molecule ranges from 1 to 4, depending on the class (e.g., IgG, IgA, and IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, and IgGA2). c One example is the disulfide-bonded dimer produced by the papain digestion of IgG. Native F c An example of an amino acid sequence is sequence number 329.

[0090] "F c Where used herein, the term "mutant" means a modified version of the native Fc but with respect to the salvage receptor F c Rn(newborn F) c This refers to a molecule or sequence that still contains the binding site of the receptor. Exemplary Fc variants and their interactions with salvage receptors are known in the art. Therefore, "F c The term "mutant" refers to non-human native F c It may include molecules or sequences that have been humanized from native F. c It includes a region that can be removed because this region confers structural features or biological activity that are not required for the antigen-binding protein of the present invention. Therefore, "F c The term "mutant" refers to one or more native F1 genes. c A missing site or residue, or one or more F c The molecule or sequence includes a modified site or residue, which affects or is involved in: (1) disulfide bond formation, (2) incompatibility with selected host cells, (3) N-terminal heterogeneity during expression in selected host cells, (4) glycosylation, (5) interaction with complement, and (6) F2 receptors other than salvage receptors. c (7) Binding to the receptor, or antibody-dependent cytotoxicity (ADCC).

[0091] In one embodiment, the Fc domain is a human IgG Fc domain, preferably derived from human IgG1, IgG2, IgG3, or IgG4, preferably derived from IgG1 or IgG2, and more preferably derived from IgG1.

[0092] In some embodiments, the antigen-binding protein is two F c Domain (F c1 and F c2 ) if it includes, for example, the TCER(registered trademark) format used in the example, these two F c The domains belong to the same antibody isotype or isotype subclass. Therefore, in some embodiments, F c1 and F c2 Both are from the IgG1 subclass, or the IgG2 subclass, or the IgG3 subclass, or the IgG4 subclass. In a preferred embodiment, F c1 and F c2 Both belong to the IgG1 subclass or the IgG2 subclass, and more preferably to the IgG1 subclass.

[0093] In some embodiments, F c The region further includes RF and / or "knob-into-hole" mutations as defined below in this specification.

[0094] "RF mutation" is generally referred to as F c This refers to mutations in the CH3 domain of the domain, specifically the amino acid HY mutations to RF, such as the H435R and Y436F mutations in the CH3 domain described by Jendeberg, L. et al., (1997, J. Immunological Meth., 201: 25-34), and is advantageous for purification purposes because it eliminates protein A binding. The antigen-binding protein has two F c If the domain is included, the RF mutation is one or both F c It may exist within a domain, preferably within one of the Fc domains.

[0095] The "knob-into-hole" technique is used to facilitate heteromultimer formation, as described in U.S. Patent No. 5,731,168 and U.S. Patent No. 8,216,805 (in particular, incorporated herein by reference), C H3 -C H3 This refers to the interface mutations T366S, L368A, and Y407V, specifically both T366S (hole) and T366W (knob). This knob-into-hole mutation can be further stabilized by introducing additional cysteine ​​amino acid substitutions Y349C and S354C.

[0096] "Knob" mutations are, for example, F149. c It exists in the amino acid sequence, and a "hole" mutation is, for example, F in SEQ ID NO: 150. c It is present in the amino acid sequence.

[0097] In some embodiments, F of one polypeptide c Domain (for example, F c1 ) is that C H3 The domain contains the amino acid substitution T366W (nob), and the other polypeptide F c Domain (for example, F c2 ) is that C H3 The domain contains amino acid substitutions T366S, L368A, and Y407V (a whole), and vice versa.

[0098] In some embodiments, F of one polypeptide c Domain (for example, F c1 ) is that C H3 The domain contains, or further contains, the amino acid substitution S354C, and the other polypeptide F c Domain (for example, F c2 ) is that C H3ド The main component contains, or further contains, the amino acid substitution Y349C, and vice versa.

[0099] Therefore, in some embodiments, F of one polypeptide c Domain (for example, F c1 ) is that C H3 The domain contains amino acid substitutions S354C and T366W (nobs), and the other polypeptide is F c Domain (for example, F c2 ) is that C H3 The domain contains amino acid substitutions Y349C, T366S, L368A, and Y407V (a whole), and vice versa.

[0100] This set of amino acid substitutions can be further extended by the inclusion of the amino acid substitution K409A in one polypeptide and F405K in the other polypeptide, as described by Wei et al. (Structural basis of a novel heterodimeric Fc for bispecific antibody production, Oncotarget. 2017). Therefore, in some embodiments, the F405K in one polypeptide can be further expanded. c Domain (for example, F c1 ) is that C H3 The domain contains, or further contains, the amino acid substitution K409A, and the other polypeptide F c Domain (for example, F c2 ) is that C H3 The domain contains, or further contains, the amino acid substitution F405K, and vice versa.

[0101] In some cases, artificially introduced cysteine ​​crosslinks can improve the stability of antigen-binding proteins, ideally without interfering with their binding properties. Such cysteine ​​crosslinks can further enhance heterodimerization.

[0102] Further amino acid substitutions (e.g., charge pair substitutions) to improve the heterodimerization of the resulting protein are described in the art (e.g., European Patent No. 2970484).

[0103] Therefore, in one embodiment, F of one polypeptide c Domain (for example, F c1 ) comprises or further comprises charge pair substitution E356K, E356R, D356R, or D356K, and D399K, or D399R, and the other polypeptide F c Domain (for example, F c2 ) includes, or further includes, charge pair substitutions R409D, R409E, K409E, or K409D, and N392D, N392E, K392E, or K392D, and vice versa.

[0104] In further embodiments, F on one or both, preferably both polypeptide chains c The domain is F c The modulo may include one or more modifications that inhibit gamma receptor (FcyR) binding. Such modifications may include L234A and L235A.

[0105] In further embodiments, F on one or both, preferably both polypeptide chains c The domain is F c The N297Q mutation may be included to remove the N-glycosylation site within the portion, and such a mutation is F c It disables the gamma receptor interaction.

[0106] "Hinge," "hinge region," or "hinge domain" is typically C H1 Domain and C H2 This refers to the flexible portion of the heavy chain located between the domains. It is approximately 25 amino acids long and is divided into an "upper hinge," an "intermediate hinge" or "core hinge," and a "lower hinge." A "hinge subdomain" refers to the upper hinge, intermediate (or core) hinge, or lower hinge. The amino acid sequences of the hinges of IgG1, IgG2, IgG3, and IgG4 molecules are shown below in this specification: IgG1:E 216 PKSCDKTHTCPPCPAPELLG (Sequence ID 330) IgG2:E 216 RKCCVECPPCPAPPVAGP (Sequence ID 331) IgG3:ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPE 216 PKSCDTPPPCPRCPAPELLG (Sequence ID 332) IgG4:E 216 SKYGPPCPSCPAPEFLG (Sequence ID 333).

[0107] In relation to the present invention, F c The amino acid positions within the domain are referred to, and these amino acid positions or residues are indicated according to the EU numbering system, as described, for example, in Edelman, GM et al., Proc. Natl. Acad. USA, 63, 78-85 (1969).

[0108] Hinge, C H2 Domain, and C H3 F consisting of domains c By incorporating a portion or part of this antigen-binding protein (especially a bispecific antigen-binding protein), Fc:Fc gamma receptor (F c The γR interaction caused the problem of nonspecific immobilization of these molecules. c γR binds to various cell surface molecules (F) with varying affinities to epitopes presented by the Fc portion of IgG molecules. c γRI, F c γRIIa, F c γRIIb, F c It is composed of γRIII). Such nonspecific (i.e., not induced by either of the two binding domains of the bispecific molecule) immobilization is undesirable due to i) its impact on the pharmacokinetics of the molecule and ii) its off-target activation of immune effector cells. c Various F for removing γR bonds cMutants and mutations have been identified. In this regard, see Morgan et al. 1995, Immunology (The N-terminal end of the C H2 domain of chimeric human IgG1 anti-HLA-DR is necessary for C1q, FcyRI and FcyRIII binding) c Loss of γRI bond, loss of C1q bond, and F c The invention discloses the exchange of residues 233-236 of human IgG1 by corresponding sequences derived from human IgG2, resulting in a reduction in γRIII binding (i.e., the absence of amino acids at residues 233P, 234V, and 235A, as well as at position 236). European Patent No. 1075496 discloses F c Antibodies and other F2 antibodies with regional diversity (e.g., missing residues or G at positions 233P, 234V, 235A, and 236, and one or more of 327G, 330S, and 331S) c The contained molecules are disclosed, in which the recombinant antibody can bind to the target molecule without initiating significant complement-dependent lysis or cell-mediated disruption of the target.

[0109] Therefore, in some embodiments, F c The region contains or further contains one or more amino acids or deletions selected from the group consisting of 233P, 234V, 235A, 236 (no residue) or G, 327G, 330S, 331S, preferably F c The region contains, or further contains, one or more amino acids selected from the group consisting of amino acids 233P, 234V, 235A, 236 (no residue) or G, and 327G, 330S, 331S, most preferably F c The region contains, or further contains, amino acids 233P, 234V, 235A, 236 (no residue), and 331S.

[0110] In a further embodiment, F cThe domain contains, or further contains, the amino acid substitution N297Q, N297G, or N297A, preferably N297Q.

[0111] The amino acid substitutions "N297Q", "N297G", or "N297A" are F c This refers to an amino acid substitution at position 297 that overrides the native N-glycosylation site within the domain. This amino acid substitution is caused by a sugar residue, as explained, for example, in Tao, MH and Morrison, SL (J Immunol. 1989 Oct 15;143(8):2595-601.). c This further prevents gamma receptor interactions and reduces the diversity of the final protein product (i.e., the antigen-binding protein of the present invention).

[0112] In a further embodiment, especially when there is no light chain, F c The domain contains or further contains the amino acid substitution C220S. The amino acid substitution "C220S" means C H1 -C L The cysteine ​​that forms the disulfide bond is missing.

[0113] In some embodiments, F c The domain contains at least two further cysteine ​​residues, or more (e.g., S354C and Y349C, or L242C and K334C), where, in order to form a heterodimer, S354C is F c1 F of one polypeptide such as c It exists within the domain, and Y349C is F c2 F of other polypeptides such as c L242C and K334C are present within the domain and / or form intradomain CC bonds in one or both polypeptides. c1 or F c2 The same F in any of the following c It is located within the domain.

[0114] The antigen-binding proteins of this disclosure may be synthetic, recombinant, isolated, manipulated, and / or purified.

[0115] When referring to polypeptides (e.g., the antigen-binding proteins of the present invention) or nucleotide sequences (e.g., nucleotide sequences encoding the antigen-binding proteins or functional fragments thereof as described herein), “purified” means that the molecules shown are present in the substantial absence of other biological macromolecules of the same type. The term “purified” means, particularly as used herein, that at least 75% by weight, 85% by weight, 95% by weight, or 98% by weight of biological macromolecules of the same type are present.

[0116] A purified nucleic acid molecule encoding a specific polypeptide refers to a nucleic acid molecule that substantially contains no other nucleic acid molecules that do not encode the polypeptide of the subject; however, this molecule may contain several additional bases or moieties that do not adversely affect the fundamental properties of its composition.

[0117] The term "isolated" means that something has been altered or removed from its natural state. For example, a nucleic acid or peptide that is naturally present in a living animal is not "isolated," but the same nucleic acid or peptide that has been partially or completely separated from coexisting material in its natural state is "isolated." Isolated nucleic acids or proteins may exist in a substantially purified form or in a non-natural environment, such as a host cell. An isolated antigen-binding protein substantially does not contain other antigen-binding proteins with different antigen specificities (for example, an antigen-binding protein that specifically binds to PRAME substantially does not contain antigen-binding proteins that specifically bind to antigens other than PRAME). Furthermore, an isolated antigen-binding protein may substantially not contain other cellular material and / or chemical substances.

[0118] A "recombinant" molecule is a molecule that has been prepared, expressed, produced, or isolated using recombinant methods. Recombinant molecules do not exist in nature.

[0119] The term "gene" refers to a DNA sequence that codes for or corresponds to a specific sequence of amino acids that constitute all or part of one or more proteins or enzymes, and may or may not include regulatory DNA sequences (e.g., promoter sequences) that determine the conditions under which this gene is expressed. Some genes that are not structural genes can be transcribed from DNA to RNA but not translated into amino acid sequences. Other genes can function as regulators of structural genes or as regulators of DNA transcription. In particular, the term "gene" may refer to genomic sequences that code for proteins (i.e., sequences containing regulators, promoters, introns, and exons).

[0120] In relation to the present invention, "affinity" is defined by the equilibrium binding between the antigen-binding protein and its antigen (i.e., the PRAME-004 peptide related to SEQ ID NO: 50 in a complex with an MHC protein). Affinity is, for example, measured by the maximum effective concentration (EC2). 50 ) or parallel dissociation constant (K D ) can be expressed as. In relation to the present invention, high affinity is K ≤100nM, ≤50nM, ≤10nM, or ≤5nM. D This refers to a connection.

[0121] "K D " is the equilibrium dissociation constant (k) between the antigen-binding protein and its antigen. off / k on (ratio of K) D And affinity are inversely correlated. K D The value is related to the concentration of antigen-binding protein, K D The lower the value, the higher the affinity of the antigen-binding protein. Affinity (i.e., K DThe value can be experimentally evaluated by various known methods, such as measuring the association and dissociation rates by surface plasmon resonance (SPR) or biolayer interferometry (BLI), as described in more detail in the "Antigen-Binding Proteins" section below in this specification. D This is preferably measured by biolayer interferometry (BLI). More preferably, the K of the antigen-binding protein for the PRAME antigen peptide. D The temperature is determined by BLI to be 20°C to 35°C, preferably 25°C to 32°C, more preferably about 30°C, and the pH is 6.5 to 8.0, preferably 7.0 to 7.6. Even more preferably, the K of the antigen-binding protein for the PRAME antigen peptide. D The optimal temperature is determined by BLI at 20°C to 35°C, preferably 25°C to 32°C, more preferably about 30°C, pH at 6.5 to 8.0, preferably 7.0 to 7.6, and salt concentration at 100 to 200 mM, preferably 120 to 175 mM, more preferably about 140 mM. Most preferably, the K of the antigen-binding protein for the PRAME antigen peptide is D The K of antigen-binding protein is determined by BLI at 30°C in a buffer consisting of PBS, 0.05% Tween-20, and 0.1% BSA. In such measurements, the concentration of antigen-binding protein is typically 1.56–500 nM, depending on the affinity of the interaction being measured. D When comparing the two peptides, the peptide-HLA loading conditions are identical for both measurements if the two peptides have similar HLA binding sensitivities, and the range of antigen-binding protein concentrations is selected considering the expected affinity.

[0122] "EC 50 The "maximum half-dose effective concentration," also known as the EC50 50 And affinity is inversely correlated, EC 50 The lower the value, the higher the affinity of the molecule. For example, "EC50 " refers to the concentration of the antigen-binding protein of the present invention that induces an intermediate response between baseline and maximum after a specific exposure time, and in particular, the concentration of the antigen-binding protein of the present invention that induces an intermediate response between baseline and maximum after a specific exposure time. EC 50 The values ​​can be experimentally evaluated using various known methods, such as binding assays like ELISA or flow cytometry, or functional assays like IFN-gamma release assays or lactate dehydrogenase (LDH) release assays.

[0123] Antigen-binding protein The antigen-binding proteins of the present invention are designed from scaffold sequences into which numerous mutations have been introduced. These antigen-binding proteins have a profile particularly suitable for therapeutic applications. Generally, identifying such antigen-binding proteins is not straightforward and typically involves a high attrition rate.

[0124] First and foremost, those skilled in the art need to identify a suitable starting (i.e., scaffold) sequence. In the present invention, this is a TCR that has good affinity (e.g., 200 μM or more) for the target peptide-HLA complex; high levels of target specificity, e.g., relatively weak or no binding to alternative peptide-HLA complexes; and can be refolded and purified in high yield. Given the degenerate nature of TCR recognition, it is extremely difficult even for those skilled in the art to determine whether a particular scaffold TCR sequence has a specificity profile that would make it suitable for design for therapeutic applications (Wooldridge, et al., J Biol Chem. 2012 Jan 6;287(2): 1 168-77).

[0125] A particularly important step is converting this TCR into a soluble format that can be stably expressed. Naturally occurring TCRs are membrane-bound and are expressed only in complex with CD3. In addition to antibodies routinely expressed as single-stranded variable fragment (scFv) molecules, the corresponding single-stranded T cell receptor variable domain (scTv) constructs are prone to aggregation and misfolding (Richman, et al. Mol Immunol. 2009 Feb;46(5):902-16. doi: 10.1016 / j.molimm.2008.09.021. Epub 2008 Oct 29). This step is essential for the production of active biological substances, but will also be indispensable for the further processing steps described below. The process of scTv conversion and the generation of stable, soluble molecules typically involves designing one or more specific mutations (e.g., substitutions, insertions, and / or deletions) in the framework region and / or CDR of the TCR starting sequence to increase scTv expression and stability. Each TCR has a different set of mutations depending on the combination of variable domains and the configuration of the CDR3. Specific mutations and / or combinations of mutations that result in a significant increase in solubility and stability are unpredictable and have a high dropout rate. In many cases, it may be impossible to achieve a significant increase in solubility and stability with a given TCR starting sequence.

[0126] The next challenge is to design TCRs that have higher affinity for target antigens while maintaining desirable properties such as specificity and yield. In nature, TCRs have weaker affinity for target antigens compared to antibodies (low micromolar range), and TCRs for cancer antigens typically have weaker antigen recognition compared to virus-specific TCRs (Aleksic, et al. Eur J Immunol. 2012 Dec;42(12):3174-9). This weak affinity, coupled with HLA downregulation in cancer cells, means that therapeutic TCRs for cancer immunotherapy typically need to be designed to increase affinity for target antigens and thus produce a more potent response. Such an increase in affinity is essential for soluble TCR-based reagents. In such cases, antigen-binding affinity in the nanomolar to picomolar range, along with a binding half-life of several hours, is desirable. The affinity maturation process typically involves designing specific mutations and / or combinations of mutations (e.g., substitutions, insertions, and / or deletions) in the CDR of a starting TCR sequence to increase the strength of antigen recognition. To significantly increase the affinity of a given TCR to a given target, a person skilled in the art may have to design combinations of mutations in the CDR from a large pool of possible options. Specific mutations and / or combinations of mutations that significantly increase affinity are unpredictable and have a high dropout rate. In many cases, it may be impossible to achieve a significant increase in affinity with a given TCR starting sequence.

[0127] The affinity maturation process must also consider the need to maintain TCR antigen specificity. Increased affinity of a TCR for its target antigen carries a significant risk of unintended cross-reactivity with other targets, resulting from the inherent degeneracy of TCR antigen recognition (Wooldridge, et al., J Biol Chem. 2012 Jan 6;287(2): 1 168-77; Wilson, et al., Mol lmmunol. 2004 Feb;40(14-15): 1047-55; Zhao ef al., J Immunol. 2007 Nov 1;179(9):5845-54). At natural affinity levels, recognition of cross-reactive antigens may be excessively low, resulting in no reaction. If cross-reactive antigens are present on normal, healthy cells, in vivo off-target binding is likely to occur, which can manifest as clinical toxicity. Therefore, in addition to increasing antigen-binding strength, those skilled in the art must also design mutations and / or combinations of mutations in the CDR that enable the TCR to retain high specificity for the target antigen and thus exhibit a favorable safety profile in preclinical trials. Again, however, the appropriate mutations and / or combinations of mutations are unpredictable. The dropout rate at this stage is even higher and, in many cases, may not be achieved at all from a given TCR starting sequence. Despite the difficulties described above, we have identified antigen-binding proteins, particularly those containing CDR-derived TCRs with high affinity (low nanomolar range) and high antigen specificity.

[0128] As a starting point, using TCR R11P3D3 disclosed in International Publication No. 2018 / 172533, incorporated herein by reference, the inventors have designed, manufactured, and tested variants of the variable alpha and beta domains of R11P3D3 in single-stranded TCR (scTCR) and TCER® formats, which may be conjugated to a Fab fragment. In this way, the inventors have identified various CDRs (in particular CDRa1, CDRa3, CDRb1, and CDR3, and as appropriate, CDRa2 and CDRb2) associated with high affinity and high specificity binding of the antigen-binding protein of the present invention to its target (i.e., the RAME-004 peptide in complex with an MHC protein).

[0129] The inventors designed a bispecific TCER® molecule, a single-stranded TCR (scTCR), and a bispecific scTCR-Fab molecule. All constructs specifically bind to peptide-MHC complexes containing the PRAME-004 peptide. The bispecific constructs disclosed in the examples further bind to effector cells (particularly T cells) via antibody-derived "recruiters." Thus, the inventors have demonstrated that CDRs can be used in single-stranded TCR constructs and bispecific TCR-antibody molecules, and have therefore demonstrated that various antigen-binding proteins with high affinity and high specificity for the PRAME-004 peptide in complex with MHC proteins can be produced using the identified CDRs.

[0130] Accordingly, in the first aspect, the present invention is an antigen-binding protein that specifically binds to a PRAME antigen peptide which contains or consists of the amino acid sequence SLLQHLIGL of SEQ ID NO: 50 and exists in complex with a major histocompatibility complex (MHC) protein, Variable domain V, including complementarity-determining regions (CDRs) CDRa1, CDRa2, and CDRa3. A And, - CDRa1 contains or consists of the amino acid sequence of VKEFQD (SEQ ID NO: 16), or an amino acid sequence different from SEQ ID NO: 16 due to one, two, or three amino acid mutations, preferably amino acid substitutions. - CDRa3 contains or consists of the amino acid sequence of ALYNNLDMR (SEQ ID NO: 33) or ALYNNYDMR (SEQ ID NO: 34), or one, two, or three (preferably one or two) amino acid mutations, preferably amino acid substitutions, that result in an amino acid sequence different from SEQ ID NO: 33 or SEQ ID NO: 34. Variable Domain V A and, Variable domain V, including CDRb1, CDRb2, and CDRb3 B And, - CDRb1 contains or consists of the amino acid sequence of SGHNS (SEQ ID NO: 10), or one, two, or three (preferably one or two) amino acid mutations, preferably amino acid substitutions, resulting in an amino acid sequence different from SEQ ID NO: 10. - CDRb3 is an amino acid sequence of ASSX1GX2X3DX4QY (wherein X1 is P, A, or T, preferably P; X2 is A or S, preferably A; X3 is T or I; X4 is T, K, or A, preferably K or A, more preferably K) (SEQ ID NO: 327), or contains an amino acid sequence different from SEQ ID NO: 327 by 1, 2, or 3 amino acid mutations, preferably amino acid substitutions. Variable Domain V B and This relates to antigen-binding proteins, including those mentioned above.

[0131] The specificity of antigen-binding proteins is determined by the amino acid sequences CDRa1, CDRa3, CDRb1, and CDRb3, and is independent of the amino acid sequences of CDRa2 and CDRb2.

[0132] In some embodiments, CDRa2 comprises or consists of the amino acid sequence of SEQ ID NO: 32, or an amino acid sequence different from SEQ ID NO: 32 by 1, 2, or 3 amino acid mutations, preferably amino acid substitutions, and / or CDRb2 comprises or consists of the amino acid sequence of SEQ ID NO: 36, or an amino acid sequence different from SEQ ID NO: 36 by 1, 2, 3, 4, 5, or 6 (preferably 5 or fewer, more preferably 4 or fewer, even more preferably 3 or fewer) amino acid mutations, preferably amino acid substitutions.

[0133] In some embodiments, the antigen-binding protein comprises CDRa1, CDRb1 as defined above, and optionally CDRa2 and CDRb2, wherein CDRa3 comprises the amino acid sequence of SEQ ID NO: 33, or an amino acid sequence different from SEQ ID NO: 33 due to one, two, or three (preferably one or two) amino acid mutations, preferably amino acid substitutions, and CDRb3 comprises the amino acid sequence ASSX1GX2X3DX4QY(SEQ ID NO: 327) (wherein X1 is P, A, or T, preferably P; X2 is A or S, preferably A; X3 is T or I; X4 is T, K, or A, preferably K or A, more preferably K), or an amino acid sequence different from SEQ ID NO: 327 due to one, two, or three amino acid mutations, preferably amino acid substitutions.

[0134] In some embodiments, the antigen-binding protein comprises CDRa1, CDRb1 as defined above, and optionally CDRa2 and CDRb2, wherein CDRa3 comprises the amino acid sequence of SEQ ID NO: 34, or an amino acid sequence different from SEQ ID NO: 34 due to one, two, or three (preferably one or two) amino acid mutations, preferably amino acid substitutions, and CDRb3 comprises the amino acid sequence ASSX1GX2X3DX4QY(SEQ ID NO: 327) (wherein X1 is P, A, or T, preferably P; X2 is A or S, preferably A; X3 is T or I; X4 is T, K, or A, more preferably K or A, preferably K), or an amino acid sequence different from SEQ ID NO: 327 due to one, two, or three amino acid mutations, preferably amino acid substitutions.

[0135] In a preferred embodiment, CDRb3 comprises the amino acid sequence ASSPGX2X3DX4QY (SEQ ID NO: 364) (wherein X2 is A or S, preferably A; X3 is T or I; X4 is T, K, or A, preferably K or A, more preferably K), or an amino acid sequence different from SEQ ID NO: 364 due to one, two, or three amino acid mutations, preferably amino acid substitutions.

[0136] In a preferred embodiment, CDRb3 comprises the amino acid sequence ASSPGX2TDX4QY (SEQ ID NO: 363) (wherein X2 is A or S, preferably A, and X4 is T, K, or A, preferably K or A, more preferably K), or an amino acid sequence different from SEQ ID NO: 363 due to one, two, or three amino acid mutations, preferably amino acid substitutions.

[0137] In other preferred embodiments, CDRb3 comprises the amino acid sequence ASSPGAX3DX4QY (SEQ ID NO: 365) (wherein X3 is T or I, preferably I, and X4 is K or A, preferably K), or an amino acid sequence different from SEQ ID NO: 365 due to one, two, or three amino acid mutations, preferably amino acid substitutions.

[0138] In some embodiments, the antigen-binding protein comprises CDRa1, CDRb1 as defined above, and optionally CDRa2 and CDRb2, wherein CDRa3 comprises the amino acid sequence of SEQ ID NO: 33, or an amino acid sequence different from SEQ ID NO: 33 due to one, two, or three (preferably one or two) amino acid mutations, preferably amino acid substitutions, and CDRb3 comprises the amino acid sequence of SEQ ID NO: 48, or an amino acid sequence different from SEQ ID NO: 48 due to one, two, three, or four (preferably one, two, or three, more preferably one or two) amino acid mutations, preferably amino acid substitutions.

[0139] In some embodiments, the antigen-binding protein comprises CDRa1, CDRb1 as defined above, and optionally CDRa2 and CDRb2, wherein CDRa3 comprises the amino acid sequence of SEQ ID NO: 33, or an amino acid sequence different from SEQ ID NO: 33 due to one, two, or three (preferably one or two) amino acid mutations, preferably amino acid substitutions, and CDRb3 comprises the amino acid sequence of SEQ ID NO: 48, or SEQ ID NO: 283, or SEQ ID NO: 281, or SEQ ID NO: 297, or an amino acid sequence different from SEQ ID NO: 48, SEQ ID NO: 297, SEQ ID NO: 281, or SEQ ID NO: 283 due to one, two, or three (preferably one or two) amino acid mutations, preferably amino acid substitutions.

[0140] In preferred embodiments, CDRa1, CDRa3, CDRb1, CDRb3, and optionally CDRa2 and CDRb2, each differ from SEQ ID NOs. 16, SEQ ID NOs. 33 or 34, SEQ ID NOs. 10, SEQ ID NOs. 327, SEQ ID NOs. 32, and SEQ ID NOs. 36 by two or fewer (preferably one or fewer) amino acid mutations, preferably amino acid substitutions. In preferred embodiments, the amino acid substitutions in the CDRs are conservative substitutions.

[0141] Variable Domain V A and variable domain V B Together, they form an antigen-binding site that binds to the PRAME-004 antigen peptide complexed with the MHC protein. Hereafter, this antigen-binding site may also be referred to as the "first antigen-binding site."

[0142] CDRa1, CDRa2, and CDRa3 are derived from the TCR α-chain variable domain, while CDRb1, CDRb2, and CDRb3 are derived from the TCR β-chain variable domain.

[0143] In addition to CDR, V A and V B This includes a framework region (FR). The FR sequence may be derived from the TCR, i.e., from the variable domains of the TCR α, β, γ, or δ chains, or from the antibody variable domain. For example, V A It contains the FR sequence of the antibody light chain variable domain, and therefore can be described as (FR1-L)-(CDRa1)-(FR2-L)-(CDRa2)-(FR3-L)-(CDRa3)-(FR4-L), V B This includes the FR sequence of the antibody heavy chain variable domain, and therefore can be described as (FR1-H)-(CDRb1)-(FR2-H)-(CDRb2)-(FR3-H)-(CDRb3)-(FR4-H). A It contains an FR sequence of an α or γ (preferably α) chain variable domain, and therefore can be described as (FR1-a)-(CDRa1)-(FR2-a)-(CDRa2)-(FR3-a)-(CDRa3)-(FR4-a), V BIt preferably contains an FR sequence of a β or δ (preferably β) chain variable domain, and therefore can be described as (FR1-b)-(CDRb1)-(FR2-b)-(CDRb2)-(FR3-b)-(CDRb3)-(FR4-b). A If the entire domain originates from the TCR α chain, then V A Also, V α It can also be called V. B If the entire domain originates from the TCR β chain, then V b Also, V β It can also be called...

[0144] In some embodiments, - The 27th position of CDRa1 according to IMGT is either V or substituted with an amino acid selected from L, I, M, F, A, T, N, Q, H, E, D, and S (in particular, selected from T, N, S, and I). - The 28th position of CDRa1 according to IMGT is either K or substituted with an amino acid selected from R, Q, H, N, A, V, S, G, L, I, and T (in particular, selected from R, A, and S). - The 38th position of CDRa1 according to IMGT is either D or substituted with an amino acid selected from E, N, Q, H, K, and R (especially N). - The 64th position of CDRa2 according to IMGT is either K or substituted with an amino acid selected from R, Q, H, N, T, V, A, L, I, M, and F (in particular, selected from R, T, and V). - The 114th position of CDRa3 according to IMGT is L or Y, or substituted with an amino acid selected from M, W, H, Q, A, I, K, R, V, D, E, F, and N (in particular, selected from H, Q, A, I, K, R, V, D, E, F, and N, and more particularly selected from H, Q, A, and I). - The 56th position of CDRb2 according to IMGT is either F or substituted with an amino acid selected from Y, M, L, W, H, V, I, and A (in particular, selected from Y, M, and L). - The 57th position of CDRb2 according to IMGT is Q, or is substituted with an amino acid selected from N, R, D, E, Q, H, K, and K (especially N), provided that if the amino acid at position 63 is T or S, then the amino acid at position 57 is not N. - The 58th position of CDRb2 according to IMGT is either N or substituted with an amino acid selected from Q, H, D, K, R, S, and T (especially S). - The 63rd position of CDRb2 according to IMGT is either T or substituted with an amino acid selected from S, V, A, D, Q, and E (in particular, selected from S and E), provided that if the amino acid at position 57 is N, then the amino acid at position 63 is neither T nor S. - The 64th position of CDRb2 according to IMGT is A, or substituted with an amino acid selected from V, L, I, S, G, and T (especially T), - The 65th position of CDRb2 according to IMGT is either V or substituted with an amino acid selected from L, I, M, A, T, F, and S (in particular, selected from I, L, and T). - The 108th position of CDRb3 according to IMGT is substituted with an amino acid that is P, A, or T, or selected from V, L, I, S, G, R, K, N, and Q (in particular, selected from R and S), provided that if the amino acid at position 110 is T or S, then the amino acid at position 108 is not N. - The 110th position of CDRb3 according to IMGT is either A or S, or substituted with an amino acid selected from V, L, I, G, T, and C (especially T), provided that if the amino acid at position 108 is N, then the amino acid at position 110 is neither T nor S. - The 113th position of CDRb3 according to IMGT is either T or I, or substituted with an amino acid selected from V, L, I, G, and T. - The 115th position of CDRb3 according to IMGT is either T, K, or A, or is substituted with an amino acid selected from G, L, I, V, R, Q, N, Y, H, E, and F (in particular, selected from L, I, V, R, Q, N, Y, H, E, and F, and more particularly selected from L, I, V, and R).

[0145] In some embodiments, - CDRa1 contains or consists of the amino acid sequence X1X2EFQX3 (SEQ ID NO: 334), where X1 is V, T, N, I, or S, preferably V, T, or N, most preferably V; X2 is K, R, S, or A, more preferably K or R, most preferably K; and X3 is D or N, preferably D. - CDRa2 contains or consists of the amino acid sequence FGPYGX1E (SEQ ID NO: 335), where X1 is K, R, T, or V, preferably K or R, and most preferably K. - CDRa3 comprises or consists of the amino acid sequence ALYNNX1DMR (SEQ ID NO: 336), where X1 is L, Y, H, Q, A, I, K, R, V, D, E, F, or N, preferably L, Y, H, Q, A, I, K, or R, more preferably L, Y, H, Q, or A, and most preferably L or Y. - CDRb1 preferably contains or consists of amino acid sequence number 10. - CDRb2 contains or consists of the amino acid sequence X1X2X3X4X5X6, where X1 is F, Y, M, or L, preferably F or Y, most preferably F; X2 is Q or N, preferably Q (if X2 is N, then X3 is also N); X3 is N or S, preferably N; X4 is T, S, or E, preferably T or S, most preferably T (if X4 is S, then X2 is Q); X5 is A or T, preferably A, X 6 is V, I, L, or T, preferably V or I, most preferably V (SEQ ID NO: 337), and more preferably CDRb2 comprises or consists of the amino acid sequence X1QX3TX5X6 (SEQ ID NO: 359), where X1 is F, Y, M, or L, preferably F or Y, most preferably F; X3 is N or S, preferably N; X5 is A or T, preferably A; and X6 is V, I, L, or T, preferably V or I, most preferably V. - CDRb3 comprises or consists of the amino acid sequence ASSX1GX2X3DX4QY (SEQ ID NO: 338), where X1 is P, R, A, T, or S, preferably P, T, or A, most preferably P; X2 is A or S, preferably A; X3 is T or I, preferably T; and X4 is K, A, L, I, V, R, Q, N, Y, T, H, E, or F, preferably K, A, L, I, V, R, Q, N, or Y, more preferably K, A, L, I, V, or R, most preferably K or A.

[0146] In some embodiments, - CDRa1 contains or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, and 28. - CDRa2 contains or consists of an amino acid sequence selected from the group consisting of the sequences of SEQ ID NO: 32, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 29. - CDRa3 contains or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 33, 34, 227, 233, 219, 229, 231, 235, 237, 221, 223, 225, and 9. - CDRb1 contains or consists of the amino acid sequence of SEQ ID NO: 10. - CDRb2 contains or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 36, 37, 38, 39, 40, 41, 42, 43, 44, and 35, and / or - CDRb3 contains or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 46, 47, 48, 49, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 273, 275, 277, 279, 281, 283, 292, 293, 294, 297, 298, 301, 302, 271, and 269.

[0147] Preferably, the antigen-binding protein includes CDRa1 of SEQ ID NO: 16, CDRa3 of SEQ ID NO: 34, CDRb1 of SEQ ID NO: 10, CDRb3 of SEQ ID NO: 48 or 292, and optionally CDRa2 of SEQ ID NO: 32 and CDRb2 of SEQ ID NO: 36.

[0148] In a preferred example, the antigen-binding protein includes CDRa1 of SEQ ID NO: 16, CDRa3 of SEQ ID NO: 34, CDRb1 of SEQ ID NO: 10, CDRb3 of SEQ ID NO: 48, and optionally CDRa2 of SEQ ID NO: 32 and CDRb2 of SEQ ID NO: 36. Therefore, the antigen-binding protein may include CDRa1 of SEQ ID NO: 16, CDRa3 of SEQ ID NO: 34, CDRb1 of SEQ ID NO: 10, CDRb3 of SEQ ID NO: 48, CDRa2 of SEQ ID NO: 32, and CDRb2 of SEQ ID NO: 36.

[0149] In another preferred example, the antigen-binding protein may include CDRa1 of SEQ ID NO: 16, CDRa3 of SEQ ID NO: 34, CDRb1 of SEQ ID NO: 10, and CDRb3 of SEQ ID NO: 292, as well as optionally CDRa2 of SEQ ID NO: 32 and CDRb2 of SEQ ID NO: 36. Thus, the antigen-binding protein may include CDRa1 of SEQ ID NO: 16, CDRa3 of SEQ ID NO: 34, CDRb1 of SEQ ID NO: 10, CDRb3 of SEQ ID NO: 292, CDRa2 of SEQ ID NO: 32, and CDRb2 of SEQ ID NO: 36.

[0150] Preferably, the antigen-binding protein includes, without modification, CDRa1 of SEQ ID NO: 16, CDRa3 of SEQ ID NO: 33, SEQ ID NO: 34, or CDRa3 of SEQ ID NO: 9, CDRb1 of SEQ ID NO: 10, CDRb3 of SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 47, SEQ ID NO: 281, SEQ ID NO: 292, SEQ ID NO: 294, SEQ ID NO: 297, SEQ ID NO: 298, SEQ ID NO: 301, or CDRb3 of SEQ ID NO: 283, and optionally CDRa2 of SEQ ID NO: 32 and CDRb2 of SEQ ID NO: 36.

[0151] In some embodiments, the PRAME antigen peptide consists of SEQ ID NO: 50.

[0152] In some embodiments, the antigen-binding protein specifically binds to the amino acid sequence of SEQ ID NO: 50 of the complex with the MHC protein.

[0153] In some embodiments, the MHC protein is an MHC class I HLA protein (e.g., HLA-A, HLA-B, or HLA-C), preferably HLA-A, and more preferably HLA-A*02.

[0154] In a preferred embodiment, the antigen-binding protein specifically binds to the structural epitope of the PRAME-004 antigen peptide of SEQ ID NO: 50. In a more preferred embodiment, the antigen-binding protein specifically binds to the functional epitope of the PRAME-004 antigen peptide of SEQ ID NO: 50.

[0155] The inventors conducted experiments to identify residues of PRAME-004 associated with binding by the antigen-binding protein of the present invention (Figure 5, Tables 4, 10, 12, 16). As a result, the inventors were able to identify amino acids 3, 5, 6, 7, and 8 of SEQ ID NO: 50 as being associated with binding. The amino acid at position 3 is strongly recognized by the antigen-binding protein of the present invention. The amino acid at position 5 is also strongly recognized. The amino acid at position 7 is the most strongly recognized. The amino acids at positions 6 and 8 have small contributions. Positions 3, 5, and 7 of SEQ ID NO: 50, as well as positions 6 and 8 as appropriate, are also referred to herein as the “binding motif” of PRAME-004. Those skilled in the art will recognize that the determination of the exact epitope or functional epitope may vary slightly depending on the method used and the cutoff value selected.

[0156] In some embodiments, the antigen-binding protein specifically binds to a functional epitope comprising or consisting of 3, 4, or 5 amino acid positions selected from the group consisting of positions 3, 5, 6, 7, and 8 of SEQ ID NO: 50, particularly positions 3, 5, and 7. In some embodiments, the antigen-binding protein specifically binds to a functional epitope comprising amino acid positions 3, 5, and 7 of SEQ ID NO: 50, or positions 3, 5, 6, and 7, or positions 3, 5, 7, and 8, or positions 3, 5, 6, 7, and 8 (preferably not amino acid positions 1 and 4 of SEQ ID NO: 50). In other words, the antigen-binding protein preferably specifically binds to amino acid positions 3, 5, and 7, and optionally positions 6 and / or 8 (preferably not positions 1 or 4) of the complex of the antigen-binding protein with an MHC protein (particularly an HLA protein, more particularly HLA-A, and even more particularly HLA-A*02) of SEQ ID NO: 50. In one embodiment, the antigen-binding protein of the present disclosure specifically binds to a functional epitope comprising at least three amino acid positions selected from the group consisting of positions 3, 5, 6, 7, and 8 of SEQ ID NO: 50, except that this antigen-binding protein does not bind to amino acid positions 1 and 4 of SEQ ID NO: 50. In one embodiment, the antigen-binding protein specifically binds to a functional epitope comprising at least six or seven amino acid positions selected from the group consisting of positions 1, 3, 4, 5, 6, 7, and 8 of SEQ ID NO: 50.

[0157] The amino acid sequence relating to SEQ ID NO: 50, in which at least one position is substituted, is referred to herein as a "PRAME variant sequence." In particular, one position is substituted with alanine (SEQ ID NOs: 318-324). Peptides having PRAME variant sequences are also referred herein as PRAME variant peptides. In one embodiment, the antigen-binding protein of the present invention has low affinity, particularly for binding to the PRAME antigen peptide of SEQ ID NO: 50. D K is ≥2, ≥5, ≥10, ≥20, or ≥30 times higher compared to DThen, it binds to a PRAME mutant peptide in which at least one of positions 1, 3, 4, 5, 6, 7, and 8 in the complex with the MHC protein, particularly one of positions 3, 5, 6, 7, and 8, and more particularly one of positions 3, 5, and 7, is substituted with alanine. Preferably, this K D This is determined as defined in the definition section above.

[0158] When used in the antigen-binding protein of the present invention, and more particularly in the bispecific antigen-binding protein, and more particularly in the TCER® format, the CDR amino acid sequence identified by the inventors increases the binding affinity, stability, and specificity of the antigen-binding protein, especially compared to the reference protein.

[0159] In this specification, "reference protein" refers to the protein on which the antigen-binding protein of the present invention is compared. The comparison between the antigen-binding protein of the present invention and the reference protein is preferably carried out in parallel under similar (preferably identical) experimental conditions. Such a reference protein may be an antigen-binding protein containing the CDR of parental / wild-type TCR R11P3D3 disclosed in International Publication No. 2018 / 172533. The reference protein is preferably in the same format as the antigen-binding protein being compared. If the antigen-binding protein is scTCR, a suitable reference protein is scTCR R11P3D3SD (SEQ ID NO: 6) containing the variable domain of TCR R11P3D3 with a stabilizing mutation. For example, the reference protein may be TCER® as described herein, containing the CDR of TCR R11P3D3. Alternatively, the reference protein may be an antigen-binding protein containing the CDR of "CDR6" [e.g., TCDR®]. The CDRs in "CDR6" are CDRa1 with amino acid sequence DRGSQS (SEQ ID NO: 339), CDRa2 with amino acid sequence IYSNGD (SEQ ID NO: 340), CDRa3 with amino acid sequence AAVIDNDQGGILT (SEQ ID NO: 341), CDRb1 with amino acid sequence PGHRA (SEQ ID NO: 342), CDRb2 with amino acid sequence YVHGEE (SEQ ID NO: 343), and CDRb3 with amino acid sequence ASSPWDSPNVQY (SEQ ID NO: 344). The reference protein may be CDR6 TCER® (TPP-1109), which comprises a first polypeptide chain containing or consisting of SEQ ID NO: 153 and a second polypeptide chain containing or consisting of SEQ ID NO: 154. TPP-1109 contains the UCHT1(V17) recruiter corresponding to SEQ ID NOs. 108 and 109. Furthermore, the reference protein may be CDR6 scTCR, which has a polypeptide chain containing or consisting of SEQ ID NO: 357. The present inventors demonstrate in examples that the antigen-binding protein containing CDR6 [particularly TCER(registered trademark) TPP-1109] binds to the amino acids at positions 5, 6, 7, and 8 of the PRAME-004 antigen peptide, but not to the amino acids at positions 2 and 3 (Table 16).Therefore, the antigen-binding protein containing CDR6 does not bind to amino acid 3 of the PRAME-004 antigen peptide, while amino acid 3, in contrast, receives strong binding / recognition from the antigen-binding protein of the present invention.

[0160] affinity The antigen-binding protein of the present invention contains a CDR sequence selected to enhance affinity for the PRAME-004:MHC complex (Figure 2, Table 3). As can be seen from the examples (Tables 4, 8, 10, 12, 15, and 16), the antigen-binding protein of the present invention exhibits high affinity (particularly for K ≤50 nM, ≤10 nM, ≤5 nM, or ≤3 nM). D ) binds to the PRAME-004:MHC complex.

[0161] Therefore, in one embodiment, the antigen-binding protein of the present invention exhibits increased affinity, particularly compared to the reference protein.

[0162] In one embodiment, the antigen-binding protein of the present invention has a K content of ≤100nM, ≤50nM, ≤10nM, preferably ≤5nM, more preferably ≤3nM, for example, 10pM~100nM, 10pM~50nM, 10pM~10nM, 10pM~5nM, 10pM~3nM. D It then binds to a complex of PRAME peptide containing or consisting of the amino acid sequence of SEQ ID NO: 50 and an HLA molecule (preferably HLA-A*02).

[0163] For example, the antigen-binding protein of the present invention is scTCR-Fab, and has K in the ranges of ≤100nM, ≤50nM, ≤40nM, ≤30nM, ≤20nM, ≤15nM, preferably ≤15nM, for example, 10pM~100nM, 10pM~50nM, 10pM~20nM, 5nM~20nM. D It then binds to a complex of PRAME peptide containing or consisting of the amino acid sequence of SEQ ID NO: 50 and an HLA molecule (preferably HLA-A*02).

[0164] In a further example, the antigen-binding protein of the present invention is TCER and has a K of ≤100nM, ≤50nM, ≤10nM, preferably ≤5nM, more preferably ≤3nM, for example 10pM~100nM, 10pM~50nM, 10pM~10nM, 10pM~5nM, 10pM~3nM D It then binds to a complex of PRAME peptide containing or consisting of the amino acid sequence of SEQ ID NO: 50 and an HLA molecule (preferably HLA-A*02).

[0165] K D Methods for measuring affinity, etc., are known to those skilled in the art, and include, for example, surface plasmon resonance (SPR) and biolayer interference. D Exemplary methods for determining this are also described in the Examples section. As is known to those skilled in the art, the experimental conditions used in these experiments (e.g., the buffer used, the concentration of the protein) can have a strong influence on the results.

[0166] Accordingly, in one example, the antigen-binding protein of the present invention is expressed as, for example, TCER® and analyzed for its binding affinity to the HLA-A*02:PRAME-004 monomer. Typically, the measurement is performed on an Octet RED384 system, for example, using settings typically recommended by the manufacturer. Briefly, the binding reaction rate was measured, typically at 30°C and at an osmotic rate of 1000 rpm using, for example, PBS, 0.05% Tween-20, and 0.1% BSA as buffer. The peptide-HLA complex (in particular the HLA-A*02 / PRAME-004 complex) was loaded onto a biosensor such as HIS1K before analyzing serial dilutions of TCER®.

[0167] As disclosed herein, the antigen-binding protein of the present invention specifically binds to a complex of a PRAME antigen peptide containing or comprising the amino acid sequence of SEQ ID NO: 50 and an HLA molecule (preferably HLA-A*02). When this antigen-binding protein is a TCR expressed on T cells, the binding of this antigen-binding protein to the complex can induce an immune response upon binding. Accordingly, in one embodiment, the antigen-binding protein of the present invention induces an immune response, preferably characterized by an increase in interferon-gamma (IFNγ) levels.

[0168] yield The inventors have demonstrated in examples that this antigen-binding protein has a high final product yield, particularly yields of ≥1 mg / l, ≥1.5 mg / l, ≥2 mg / l, ≥5 mg / l, ≥10 mg / l, ≥15 mg / l, ≥20 mg / l, ≥30 mg / l, ≥40 mg / l, ≥50 mg / l, and ≥60 mg / l (Tables 5, 6, 7, 9, 11, and 14).

[0169] The inventors have demonstrated in examples that this antigen-binding protein has a high final product yield, in particular an increased yield compared to the reference protein, and more specifically an increased yield compared to the antigen-binding protein containing the CDR of "CDR6" expressed under the same conditions (Tables 5, 6, 7, 9, 11, and 14).

[0170] For example, the antigen-binding protein is scTCR-Fab (described in Example 2) and has product yields of ≥8 mg / l, ≥10 mg / l, ≥15 mg / l, ≥20 mg / l, ≥30 mg / l, ≥40 mg / l, ≥50 mg / l, ≥60 mg / l, ≥70 mg / l, for example, 8 mg / l to 85 mg / l, 10 mg / l to 85 mg / l, 14 mg / l to 85 mg / l, and 50 mg / l to 85 mg / l.

[0171] Alternatively, this antigen-binding protein is a TCER containing VL and VH of Recruiter UCHT1V17, and has a product yield of ≥10 mg / l, ≥12 mg / l, ≥15 mg / l, ≥16 mg / l, ≥17 mg / l, ≥18 mg / l, preferably ≥15 mg / l, for example, 10 mg / l to 30 mg / l, 15 mg / l to 25 mg / l, 15 mg / l to 30 mg / l, preferably 15 mg / l to 30 mg / l.

[0172] The final product yield is typically determined 10–11 days after transfection. Methods for measuring product yield are known to those skilled in the art. Exemplary procedures are described in the Examples section.

[0173] Therefore, in one embodiment, the antigen-binding protein of the present invention exhibits improved yield, particularly compared to the reference protein, when expressed under the same conditions.

[0174] stability The inventors have demonstrated in examples that this antigen-binding protein has high stability (Tables 5, 6, 7, 9, 11, and 14).

[0175] The term “stability” in relation to the present invention refers to physical stability and can be qualitatively and / or quantitatively evaluated using various analytical techniques described in the art, for example, Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, NY, Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993). To measure stability, a sample containing the antigen-binding protein of the present invention may be tested in a stability test, in which the sample is exposed to stress conditions for a selected period, followed by quantitative analysis of chemical and physical stability, and, where appropriate, qualitative analysis, using appropriate analytical techniques. In relation to the present invention, this method particularly refers to the evaluation of aggregate formation by measuring turbidity [e.g., dynamic light scattering (DLS) or light shielding (LO)] and / or by visual inspection (e.g., by determining color and clarity) [e.g., using size exclusion chromatography (SEC)]. A sample is considered stable if it has only slight aggregates as defined below.

[0176] In relation to the present invention, improved stability refers, for example, to increased physical stability when exposed to heat stress. Therefore, the newly developed antigen-binding protein of the present invention can withstand stress conditions (particularly heat stress) better than the reference protein.

[0177] "Low aggregation" means, for example, that a sample containing the antigen-binding protein typically has monomer content of ≥80%, ≥85%, ≥90%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, and ≥99% (e.g., monomer content of 94%-99%, 95%-99%, 96%-99%, and 97%-99%) after exposure to stress conditions (e.g., 14 days at 40°C in a buffer such as PBS) when measured by SEC such as SEC-HPLC in a buffer such as PBS. In the case of SEC, a difference of 1%, 2%, 3%, 4%, preferably 1 or 2%, more preferably 1% of monomer content is considered a significant difference in relation to the present invention under test conditions that depend on the column used, operating pressure, and buffer rate. In other words, if the reference antigen-binding protein has a monomer content of 96% and the antigen-binding protein of the present invention has a monomer content of 97%, then the monomer content of the antigen-binding protein of the present invention is significantly different from that of the reference antigen-binding protein when measured under the same conditions, and therefore significantly increased.

[0178] The inventors have demonstrated that this antigen-binding protein is stable and, in particular, has monomer content of ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, and ≥97% after 14 days at 40°C in a buffer such as PBS (Tables 5, 6, 7, 9, 11, and 14).

[0179] In one embodiment, the antigen-binding protein of the present invention is stable or has improved stability, particularly compared to a reference protein, when exposed to stress conditions over a specific period of time (for example, exposed to a temperature of 40°C for 14 days).

[0180] In one embodiment, the antigen-binding protein of the present invention, when exposed to stress conditions over a specific period of time (for example, exposure to a temperature of 40°C for 14 days), shows no aggregation, only slight aggregation, or a reduction in aggregation, particularly compared to a reference protein.

[0181] specificity In examples (Figures 3-6, Tables 3, 4, 8, 13, 15), the inventors have demonstrated that the antigen-binding protein of the present invention binds with high specificity to the PRAME antigen peptide containing SEQ ID NO: 50 in a complex with the target antigen, i.e., a complex with an MHC protein (preferably a complex with HLA-A*02).

[0182] As described above, the inventors identified amino acid positions 3, 5, 6, 7, and 8 of Sequence ID No. 50, particularly positions 3, 5, and 7, which are related to the binding of the antigen-binding protein of the present invention to the PRAME-004 antigen peptide (i.e., the "binding motif" of the PRAME-004 antigen peptide). The inventors identified, for example, potential off-target peptides that are similar to the sequence and / or motif of PRAME-004 and therefore increase the risk of the antigen-binding protein binding to PRAME-004.

[0183] In relation to the present invention, “analogous peptide” as used herein refers to a potential off-target peptide, i.e., a peptide (e.g., homologous sequence or similar motif) to which the antigen-binding molecule of the present invention may bind based on its biochemical / biophysical properties. Analogous peptides are typically 8 to 11 amino acids long. Analogous peptides related to the present invention are typically presented by MHC. Furthermore, examples of analogous peptides related to the present invention include peptides containing or consisting of an amino acid sequence similar to the amino acid sequence of the PRAME-004 antigen peptide, and more particularly, peptides containing an epitope in which some or all amino acids have the same and / or similar biochemical / biophysical properties as the amino acids constituting the epitope of the PRAME-004 antigen peptide, compared to the epitope of the PRAME-004 antigen peptide. In some embodiments, similar peptides investigated in connection with the present invention were selected from a database of HLA-A*02 binding peptides presented by tumors and normal tissues (XPRESIDENT® database) using similarity scoring within the binding-related sites of PRAME-004 and the requirement of detection of at least one in normal tissue. Binding of antigen-binding proteins to similar peptides presented by MHC proteins may cause adverse reactions. Such adverse reactions may be “off-tumor” side effects, such as cross-reactivity between certain TCRs and similar peptides in healthy tissue, as reported in Lowdell et al., Cytotherapy, published on December 4, 2018.

[0184] In particular, the following peptides are analogous peptides related to the present invention: TMED9-001 (SEQ ID NO: 51), CAT-001 (SEQ ID NO: 52), DDX60L-001 (SEQ ID NO: 53), LRRC70-001 (SEQ ID NO: 54), PTPLB-001 (SEQ ID NO: 55), HDAC5-001 (SEQ ID NO: 56), VPS13B-002 (SEQ ID NO: 57), ZNF318-001 (SEQ ID NO: 58), CCDC51-001 (SEQ ID NO: 59), IFT17-003 (SEQ ID NO: 60), DIAPH1-004 (SEQ ID NO: 62), FADS2-001 (SEQ ID NO: 63), FRYL-0 03 (SEQ ID NO: 64), GIMAP8-001 (SEQ ID NO: 65), HSF1-001 (SEQ ID NO: 66), KNT-001 (SEQ ID NO: 67), MAU-001 (SEQ ID NO: 68), MCM4-001 (SEQ ID NO: 69), MPPE1-001 (SEQ ID NO: 71), MYO1B-002 (SEQ ID NO: 72), PRR12-001 (SEQ ID NO: 73), PTRF-003 (SEQ ID NO: 74), RASGRP1-001 (SEQ ID NO: 75), SMARCD1-001 (SEQ ID NO: 76), TGM2-001 (SEQ ID NO: 77), VAV1-001 (SEQ ID NO: 78), VIM-009 (SEQ ID NO: 317)FARSA-001 (SEQ ID NO: 306), ALOX15B-003 (SEQ ID NO: 304), FAM114A2-002 (SEQ ID NO: 305), GPR56-002 (SEQ ID NO: 307), IGHD-002 (SEQ ID NO: 308), NOMAP-3-0972 (SEQ ID NO: 309), NOMAP-3-1265 (SEQ ID NO: 310), NOMAP-3-1408 (SEQ ID NO: 311), NOMAP-3-1587 (SEQ ID NO: 312), NOMAP-3-1768 (SEQ ID NO: 313), NOMAP-5-0765 (SEQ ID NO: 314), PDCD10-004 (SEQ ID NO: 315), TSN-001 (SEQ ID NO: 316), ARMC9-002 (SEQ ID NO: 187), CLI-001 (SEQ ID NO: 188), COPG1-001 (SEQ ID NO: 190), COPS7A-001 (SEQ ID NO: 192), EIF-009 (SEQ ID NO: 194), EXT2-006 (SEQ ID NO: 196), LMNA-001 (SEQ ID NO: 198), PKM-005 (SEQ ID NO: 200), PSMB3-002 (SEQ ID NO: 202), RPL-007 (SEQ ID NO: 204), SPATS2L-003 (SEQ ID NO: 206), SYNE1-002 (SEQ ID NO: 208), TGM2-002 (SEQ ID NO: 210), and TPR-004 (SEQ ID NO: 212).

[0185] The term "specificity" generally refers to the ability of an antigen-binding protein to distinguish the target peptide from the similar peptides defined above. In other words, the antigen-binding protein binds to the PRAME-004:MHC complex with high affinity, particularly to K molecules less than 100 nM, less than 50 nM, less than 10 nM, preferably less than 5 nM. D It binds to the MHC complex, but does not significantly bind to the similar peptide:MHC complex.

[0186] Those skilled in the art will recognize that some similar peptides will not be bound to the antigen-binding protein of the present invention to a detectable extent (for example, peptides in which binding signals or functional reactions above background levels are undetectable), where “background levels” refers to binding signals or functional reactions observed in the case of non-homologous “dissimilar” peptides or observed in the absence of the peptide.

[0187] In the case of other similar peptides, very low but insignificant binding is detectable. This latter similar peptide can also be described as a “potentially relevant” similar peptide. The expressions “insignificant binding” and “not binding significantly” imply that the antigen-binding protein, 1) PRAME-004 peptide: K related to binding to MHC complex D Compared to that, K increased by ≥25, ≥30, ≥40, ≥50, ≥75, or ≥100 times. D Binding (for example, by binding to an analogous peptide: MHC complex); 2) Showing a significantly reduced "functional response" (e.g., a functional response to an analog peptide-MHC complex) compared to the PRAME-004 peptide-MHC complex; or 3) Compared to detection by PRAME-004 peptide:MHC polymer, detection by labeled similar peptide:MHC polymer should be significantly reduced. This indicates.

[0188] Affinity (especially K D ) is preferably measured using biolayer interferometry (BLI) as described in the Examples section. PRAME-004 peptide: K related to binding to MHC complex D Similar peptides compared to K regarding binding to MHC complexes D The increase of also two K D It can also be expressed as a ratio of . For example, the K ratio of analogous peptides to MHC complexes. D However, regarding K for binding of PRAME-004 peptide to the MHC complex... D If it has increased 100 times compared to, then K D The ratio "analogous peptide / PRAME-004" is 100. Those skilled in the art will recognize that the affinity of the analogous peptide to the MHC complex may not be measurable if the binding is too weak.

[0189] "Functional response" refers to a response measured by a functional assay (e.g., an activation assay such as an IFN-gamma release assay) or a cytotoxic assay such as an LDH release assay as described in the experimental section below. An IFN-gamma release assay measures IFN-gamma released by T cells exposed to a specific peptide:MHC complex. An LDH release assay measures LDH released from target cells expressing a peptide:MHC complex on their surface, which is then killed by T cells that specifically bind to this peptide:MHC complex. This binding may be direct via a TCR expressed on the T cell, or indirect via a soluble bispecific molecule that binds to both the peptide:MHC complex and the T cell (i.e., recruits the T cell). PRAME-004 EC regarding binding to peptide:MHC complex 50 In comparison, EC 50 If the value increases by ≥25, ≥30, ≥40, ≥50, ≥75, or ≥100 times, preferably by ≥200, ≥300, ≥500, or ≥1000 times, the functional response in the IFN-gamma release assay is considered to have significantly decreased. PRAME-004 peptide: EC regarding binding to MHC complex 50 In comparison, EC 50 If the value increases by ≥25, ≥30, ≥40, ≥50, ≥75, or ≥100 times, and preferably by ≥200, ≥300, ≥500, or ≥1000 times, the functional response in the LDH release assay is considered to be significantly reduced.

[0190] Detection by labeled analogous peptide:MHC multimer refers specifically to staining with analogous peptide:MHC tetramer, where the antigen-binding protein is expressed on the surface of cells (preferably yeast cells) (Examples 1.1 and 1.2). If the number of positive (i.e., stained) cells is ≤5%, ≤3%, or ≤1% of the total number of cells, or if the number of positive cells is ≤10%, ≤5%, or ≤2.5% of the positive cells stained with PRAME-004 peptide:MHC tetramer, or EC 50However, if the detection rate decreases to less than 1 / 50, less than 1 / 75, less than 1 / 100, less than 1 / 150, or less than 1 / 200, the detection rate is considered to have decreased significantly.

[0191] The antigen-binding protein of the present invention is designed to have high affinity for the target peptide and to avoid binding to similar peptides. This is a significant advantage of the antigen-binding protein of the present invention because binding to similar peptides increases the risk of side effects when present in normal tissue. Therefore, the fact that the antigen-binding protein of the present invention binds to similar peptides only with low affinity makes this antigen-binding protein a promising anti-cancer treatment in terms of safety.

[0192] The inventors have demonstrated that antigen-binding proteins (particularly the TCER® molecule) induce cytolysis in T2 cells loaded with the target peptide PRAME-004, as measured by an LDH release assay (Table 17). The inventors have further demonstrated that antigen-binding proteins (particularly the TCER® molecule) induce cytolysis in PRAME-positive tumor cell lines, as measured by an LDH release assay, but PRAME-negative tumor cell lines are unaffected by co-culture with the TCER® molecule (Figures 7-9). These in vitro experiments further demonstrate the safety of the antigen-binding protein of the present invention and confirm that its cytotoxic effect is highly selective for PRAME-positive tumor tissue. Therefore, the molecule of the present invention exhibits a beneficial safety profile.

[0193] In some embodiments, the antigen-binding proteins of the present invention are TMED9-001, CAT-001, DDX60L-001, LRRC70-001, PTPLB-001, HDAC5-001, VPS13B-002, ZNF318-001, CCDC51-001, IFT17-003, DIAPH1-004, FADS2-001, FRYL-00 in complex with MHC. 3, GIMAP8-001, HSF1-001, KNT-001, MAU-001, MCM4-001, MPPE1-001, MYO1B-002, PRR12-001, PTRF-00 3, RASGRP1-001, SMARCD1-001, TGM2-001, VAV1-001, VIM-009, FARSA-001, ALOX15B-003, FAM114A2-0 It does not significantly bind to at least one, at least two, at least three, at least four, at least five, at least ten, at least 20, or all of the similar peptides selected from the group consisting of 02, GPR56-002, IGHD-002, NOMAP-3-0972, NOMAP-3-1265, NOMAP-3-1408, NOMAP-3-1587, NOMAP-3-1768, NOMAP-5-0765, PDCD10-004, TSN-001, ARMC9-002, CLI-001, COPG1-001, COPS7A-001, EIF-009, EXT2-006, LMNA-001, PKM-005, PSMB3-002, RPL-007, SPATS2L-003, SYNE1-002, TGM2-002, and TPR-004.

[0194] In preferred embodiments, the antigen-binding protein of the present invention does not significantly bind to IFT17-003 in its complex with MHC.

[0195] In some embodiments, the antigen-binding proteins of the present invention, compared to the functional response to PRAME-004 peptide:MHC complex, are TMED9-001, CAT-001, DDX60L-001, LRRC70-001, PTPLB-001, HDAC5-001, VPS13B-002, ZNF318-001, CCDC51-001, IFT17-003, DIAP in complex with MHC. H1-004, FADS2-001, FRYL-003, GIMAP8-001, HSF1-001, KNT-001, MAU-001, MCM4-001, MPPE1-001, MYO1B-00 2, PRR12-001, PTRF-003, RASGRP1-001, SMARCD1-001, TGM2-001, VAV1-001, VIM-009, FARSA-001, ALOX15B-0 03, FAM114A2-002, GPR56-002, IGHD-002, NOMAP-3-0972, NOMAP-3-1265, NOMAP-3-1408, NOMAP-3-1587, NO MAP-3-1768, NOMAP-5-0765, PDCD10-004, TSN-001, ARMC9-002, CLI-001, COPG1-001, COPS7A-001, EIF-009, The antigen-binding protein of the present invention exhibits a significantly lower functional response to at least one, at least two, at least three, at least four, at least five, at least ten, at least 20, or all of the analogous peptides selected from the group consisting of EXT2-006, LMNA-001, PKM-005, PSMB3-002, RPL-007, SPATS2L-003, SYNE1-002, TGM2-002, and TPR-004. In a preferred embodiment, the antigen-binding protein of the present invention exhibits a significantly lower functional response to IFT17-003 in complex with MHC compared to its functional response to the PRAME-004 peptide:MHC complex.

[0196] In some embodiments, the antigen-binding protein of the present invention (in particular, a soluble bispecific antigen-binding protein, more particularly, an antigen-binding protein in TCER® format) has a K factor related to binding to the PRAME-004 peptide:MHC complex. DK is increased by ≥25, ≥30, ≥40, ≥50, ≥75, or ≥100 times compared to DAnd in the complex with MHC, TMED9-001, CAT-001, DDX60L-001, LRRC70-001, PTPLB-001, HDAC5-001, VPS13B-002, ZNF318-001, CCDC51-001, IFT17-003, DIAPH1-004, FADS2-001, FRYL-003, GIMAP8-001, HSF1-001, KNT-001, MAU-001, MCM4-001, MPPE1-001, MYO1B-002, PRR12-001, PTRF-003, RASGRP1-001, SMARCD1- 001, TGM2-001, VAV1-001, VIM-009, FARSA-001, ALOX15B-003, FAM114A2-002, GPR56-002, IGHD-002, NOMAP-3-0972, NOMAP-3-1265, NOMAP-3-1408, N OMAP-3-1587, NOMAP-3-1768, NOMAP-5-0765, PDCD10-004, TSN-001, ARMC9-002, CLI-001, COPG1-001, COPS7A-001, EIF-009, EXT2-006, LMNA-001, PKM Selected from the group consisting of -005, PSMB3-002, RPL-007, SPATS2L-003, SYNE1-002, TGM2-002, and TPR-004, in particular GIMAP8-001, MYO1B-002, SMARCD1-001, VIM-009, FARSA-001, ALOX15B-003, FAM114A2-002, GPR56-002, IGHD-002, NOMAP-3-0972, NOMAP-3-1265, NOMAP-3-1408, NOMAP-3-1587, NOMAP-3-1768, NOMAP-5-07 It binds to at least one, at least two, at least three, at least four, at least five, at least ten, at least fifteen, or all of the analogous peptides selected from the group consisting of 65, PDCD10-004, TSN-001, and / or the group consisting of ARMC9-002, CLI-001, COPG1-001, COPS7A-001, EIF-009, EXT2-006, LMNA-001, PKM-005, PSMB3-002, RPL-007, SPATS2L-003, SYNE1-002, TGM2-002, and TPR-004.In preferred embodiments, the antigen-binding protein of the present invention (in particular, a soluble bispecific antigen-binding protein, more particularly, an antigen-binding protein in TCER® format) has a K factor related to binding to the PRAME-004 peptide:MHC complex. D K is increased by ≥25, ≥30, ≥40, ≥50, ≥75, or ≥100 times compared to D Then, it binds to IFT17-003 in the complex with MHC.

[0197] In some embodiments, the antigen-binding proteins of the present invention, particularly when expressed on the cell surface, and more particularly when expressed on the yeast cell surface, are detected in complex with MHC compared to detection by PRAME-004 peptide:MHC multimer, including TMED9-001, CAT-001, DDX60L-001, LRRC70-001, PTPLB-001, HDAC5-001, VPS13B-002, ZNF318-001, CCDC51-001, IFT17-003, DIAPH1-004, FADS2-001, FRYL-003, G IMAP8-001, HSF1-001, KNT-001, MAU-001, MCM4-001, MPPE1-001, MYO1B-002, PRR12-001, PTRF-003, RASGRP1-001, SMARCD1-001, TGM2-001, VAV1- 001, VIM-009, FARSA-001, ALOX15B-003, FAM114A2-002, GPR56-002, IGHD-002, NOMAP-3-0972, NOMAP-3-1265, NOMAP-3-1408, NOMAP-3-1587, NOMA This group includes similar peptides selected from the group consisting of P-3-1768, NOMAP-5-0765, PDCD10-004, TSN-001, ARMC9-002, CLI-001, COPG1-001, COPS7A-001, EIF-009, EXT2-006, LMNA-001, PKM-005, PSMB3-002, RPL-007, SPATS2L-003, SYNE1-002, TGM2-002, and TPR-004, and in particular TMED9-001, CAT-001, DDX60L-001, LRRC70-001, and PTPLB. The group comprises similar peptides selected from the group consisting of -001, HDAC5-001, VPS13B-002, ZNF318-001, CCDC51-001, IFT17-003, DIAPH1-004, FADS2-001, FRYL-003, GIMAP8-001, HSF1-001, KNT-001, MAU-001, MCM4-001, MPPE1-001, MYO1B-002, PRR12-001, PTRF-003, RASGRP1-001, SMARCD1-001, TGM2-001, and VAV1-001, and more particularly,Labeled similar peptides containing IFT17-003 show significantly lower detection by MHC polymers.

[0198] The antigen-binding protein of the present invention has a high safety profile.

[0199] In this specification, "safety profile" refers to the ability to distinguish between tumor cells (particularly PRAME-004:MHC complex-presenting tumor cells) and healthy cells. This ability is often represented by the display of a safety window.

[0200] "Safety window" or "therapeutic window" as used herein refers to a parameter that compares the concentration of a compound required to induce a certain degree of cytotoxicity (e.g., 10%, 50%, 90%, or 100% cytotoxicity) in tumor cells (particularly PRAME-004:MHC complex-presenting tumor cells) with the concentration required to induce cytotoxicity (preferably a similar degree of cytotoxicity, more preferably a similar degree of cytotoxicity) in healthy cells. For example, if the concentration of antigen protein required to induce 90% cytotoxicity in tumor cell lines is 1 pM, and the concentration required to induce 90% cytotoxicity in healthy cells is 1000 pM, then the safety window is 1000 because the cytotoxic concentration required for tumor cell lines is 1 / 1000th of the concentration required for healthy cells.

[0201] In some embodiments, the safety window is defined as the maximum half-volume (50%) cytotoxicity (EC) against tumor cells. 50 The concentration of the compound required to induce 50% cytotoxicity (EC) in healthy cells, and the maximum 50% cytotoxicity (EC) 50 The concentration of the compound required to induce ) is compared with that of the tumor cell line. As a result, with respect to antigen-binding proteins, EC 50 This is 1 pM, and for example, the EC ratio for healthy cells 50 If the EC is 1000 pM, the safety window is 1000 because EC is a tumor cell line. 50This is because it is 1 / 1000th of what it would be in healthy cells.

[0202] In preferred embodiments, the antigen-binding protein of the present invention is characterized by a safety window of ≥100, ≥500, ≥1000, ≥2000, ≥3000, ≥4000, ≥5000, ≥6000, ≥8000, ≥10000, for example, 500 to 10000, preferably 1000 to 10000.

[0203] In this specification, “PRAME-004:MHC complex-presenting cells” refer to cells that present the PRAME antigen peptide in a complex with an MHC molecule on their surface, and the copy number of the PRAME-004:MHC complex can be determined by methods known to those skilled in the art. In a preferred embodiment, the PRAME-004:MHC complex-presenting cells are tumor cells, which are preferably cancers as defined in the “Therapeutics and Uses” section below. In relation to the present invention, the PRAME-004:MHC complex is excessively presented on the cell surface of PRAME-004:MHC complex-presenting cells compared to the level of the complex on the surface of cells in normal (healthy) tissue (also referred to as “healthy cells”). “Excessively presented” means that the PRAME-004:MHC complex is present at a level at least 1.2 times the level present in healthy tissue, preferably at a level at least 2 times, more preferably 5 to 10 times, the level present in healthy tissue or cells.

[0204] In one embodiment, PRAME-004:MHC complex-presenting cells have PRAME-004:MHC complex copy numbers greater than 50, greater than 80, greater than 100, greater than 120, greater than 150, greater than 300, greater than 400, greater than 600, greater than 800, greater than 1000, greater than 1500, and greater than 2000, preferably having PRAME-004:MHC copy numbers of 50 to 2000, for example, 80 to 2000, for example, 100 to 2000, for example, 120 to 2000.

[0205] "Copy number" as used herein refers to the number of PRAME-004:MHC complexes, as defined in relation to the present invention, present on the cell surface of a cell (e.g., PRAME-004:MHC-presenting cell, e.g., cancer cell, or healthy cell). The copy number of a protein can be determined by a variety of methods known in the art, including FACS analysis of diseased cells with a fluorescently labeled antigen-binding protein.

[0206] "Healthy cells" or "normal tissue cells" as used herein refer to cells that are not tumor cells, and preferably, healthy cells refer to tissue cells surrounding PRAME-004:MHC-presenting cells, and more particularly, tissue cells surrounding PRAME-004:MHC complex-presenting tumor cells. However, in some cases, healthy cells may also express and present the PRAME-004:MHC complex on their surface. Typically, in healthy cells relating to the present invention, as will be understood to those skilled in the art, the PRAME-004:MHC complex is present in smaller amounts (copy numbers) compared to tumor cells. Thus, in one embodiment, healthy cells have PRAME-004:MHC complex copy numbers less than 50, less than 20, or less than 10, preferably less than 10, and preferably between 0 and 10.

[0207] Healthy cells are preferably selected from the group consisting of: astrocytes, GABAergic neurons, cardiomyocytes, cardiac microvascular endothelial cells, chondrocytes, aortic endothelial cells, coronary artery endothelial cells, dermal microvascular endothelial cells, mesenchymal stem cells, nasal epithelial cells, peripheral blood mononuclear cells, pulmonary artery smooth muscle cells, pulmonary fibroblasts, epidermal keratinocytes, renal cortical epithelial cells, and tracheal smooth muscle cells, preferably astrocytes, in particular iPSC-derived astrocytes, cardiomyocytes, in particular iPSC-derived cardiomyocytes, aortic endothelial cells, mesenchymal stem cells, and tracheal smooth muscle cells.

[0208] In one preferred embodiment, the concentration of the antigen-binding protein of the present invention required to achieve at least 90%, preferably 100%, cytotoxicity in tumor cells (particularly PRAME-004:MHC complex-presenting tumor cells) is such that it is used in astrocytes, GABAergic neurons, cardiomyocytes, cardiac microvascular endothelial cells, chondrocytes, aortic endothelial cells, coronary artery endothelial cells, dermal microvascular endothelial cells, mesenchymal stem cells, nasal epithelial cells, peripheral blood mononuclear cells, pulmonary artery smooth muscle cells, pulmonary fibroblasts, epidermal keratinocytes, renal cortical epithelial cells, and tracheal smooth muscle The concentration is less than 1 / 100, less than 1 / 500, less than 1 / 10000, less than 1 / 500, less than 1 / 10000, less than 1 / 500, less than 1 / 10000, compared to the concentration required to achieve at least 10%, at least 50%, at least 90%, or 100% cytotoxicity in healthy cells, preferably astrocytes, in particular iPSC-derived astrocytes, cardiomyocytes, in particular iPSC-derived astrocytes, iPSC-derived cardiomyocytes, aortic endothelial cells, mesenchymal stem cells, and tracheal smooth muscle cells.

[0209] The inventors have demonstrated that the CDRs defined in these claims can be used with antigen-binding proteins having various formats. For example, in the experimental section, the inventors used these CDRs with single-stranded TCR constructs, such as TCER® molecules and bispecific TCRs including scTCRs fused with Fab fragments (scTCR-Fab).

[0210] Therefore, those skilled in the art will understand from these experiments that the CDRs described herein can be used in various antigen-binding proteins of the present invention.

[0211] In one embodiment, the epitope and binding properties are preserved even if the format of the antigen-binding protein is changed.

[0212] In some embodiments, the antigen-binding protein is a TCR or an antibody. Those skilled in the art will recognize that when the antigen-binding protein is an antibody, this “antibody” includes at least the sequences of CDR1, CDR3, and optionally CDR2 derived from the TCR as defined in these claims, and is therefore neither a native antibody nor a conventional antibody. However, an antigen-binding protein including, for example, a CDR derived from a TCR, and a framework region derived from an antibody, and a constant domain derived from an antibody, will have the overall structure of a conventional antibody and may be referred to as an “antibody.”

[0213] In some embodiments, the antigen-binding protein is bispecific, and in particular, a bispecific TCR, a bispecific antibody, or a bispecific TCR antibody molecule. Those skilled in the art will recognize that even when the antigen-binding protein is a bispecific “antibody,” one of the antigen-binding sites may include sequences of CDR1, CDR3, and optionally CDR2 derived from the TCR as defined in these claims, and the other antigen-binding site may be entirely antibody-derived.

[0214] In one embodiment, the antigen-binding protein is understood to be of human origin, generated from a human antigen locus, and therefore to contain a human sequence (in particular, a human TCR or antibody sequence).

[0215] In one embodiment, the antigen-binding protein is characterized as an affinity-mature antigen-binding protein, which is capable of specifically binding the PRAME-004 antigen peptide (particularly the PRAME-004:MHC complex) with higher affinity than the parent molecule (particularly TCR R11P3D3).

[0216] In some embodiments, the antigen-binding protein is V A A first polypeptide chain containing and V B It includes a second polypeptide chain containing the following.

[0217] In some embodiments, the first and second polypeptides, therefore V A and V BIt is located on a single polypeptide chain. Such a single-chain construct may be a single-chain TCR (scTCR), a single-chain antibody, or a single-chain bispecific antigen-binding protein, and in particular may be a single-chain bispecific antibody, a single-chain bispecific TCR, or a single-chain bispecific TCR antibody molecule. An example of a single-chain TCR (scTCR) is the construct used in Example 1, which may also be referred to as a single-chain TCR variable domain ("scTv") molecule. An example of a single-chain "antibody" would be scFv, in which the CDR is replaced by a CDR derived from the TCR. An example of a single-chain bispecific antibody would be an antibody in which one binding site is antibody-derived and the other binding site is TCR-derived or contains at least a TCR-derived CDR. As discussed above, such a hybrid antigen-binding protein may also be referred to as a single-chain bispecific TCR or a single-chain bispecific TCR antibody molecule.

[0218] Framework domain The inventors of this invention have further discovered that specific mutations in the framework region of the antigen-binding protein have a favorable effect compared to the parental TCR R11P3D3.

[0219] V A The advantageous mutations are as follows: - N20K (removes naturally occurring possible N-glycosylation sites), - W44K(V B In combination with Q44E, it improves the matching, affinity, and stability of the variable domain), and - A52F, V55Y, K92T, and G93D (increase the stability of antigen-binding proteins).

[0220] V B The advantageous mutations are as follows: - A84D, A84E, A84Q, A84N, A84S, preferably A84D (increases affinity for peptide-MC complex) - Q44E(V A(When combined with W44K, it improves the matching, affinity, and stability of variable domains.) - M46P and R48Q (increase the stability of antigen-binding proteins).

[0221] Mutations are indicated according to IMGT nomenclature.

[0222] Therefore, the antigen-binding protein of the present invention is preferably V A (V of R11P3D3) α (compared to) containing one or more (preferably all) of N20K, W44K, A52F, V55Y, K92T, and G93D, and V B (V of R11P3D3) β (Comparison with) includes one or more (preferably all) of A84D, Q44E, M46P, and R48Q.

[0223] The antigen-binding protein of the present invention is V A (V of R11P3D3) α (compared to) may further include one or more of L2M, L39I, and Q14K, and V B (V of R11P3D3) β (Comparison with) may further include one or more of E11L, E11K, and R22H.

[0224] Therefore, the antigen-binding protein of the present invention is preferably V A It contains one or more (preferably all) of the following amino acids: 20K, 44K, 52F, 55Y, 92T, and 93D, and V B It contains one or more (preferably all) of 84D, 44E, 46P, and 48Q.

[0225] The antigen-binding protein of the present invention is V A It may further contain one or more of the following amino acids 2M, 39I, and 14K, and V B It may further contain one or more of 11L or 11K and 22H.

[0226] In one embodiment, V A This further includes one or more framework regions selected from the group consisting of FR1-a, FR2-a, FR3-a, and FR4-a, preferably all of the framework regions. - FR1-a contains or comprises the amino acid sequence of SEQ ID NO: 345 or SEQ ID NO: 346, or an amino acid sequence that is at least 85%, 90%, or 95% identical to SEQ ID NO: 345, preferably containing K or N at position 20, more preferably containing K, and / or containing L or M at position 2; - FR2-a contains or comprises the amino acid sequence of SEQ ID NO: 347 or SEQ ID NO: 348, or an amino acid sequence that is at least 85%, 90%, or 95% identical to SEQ ID NO: 347, preferably containing L, I, or M at position 39, more preferably L or I; containing A or D at position 47, more preferably A; containing K or W at position 44, preferably K; containing F or A at position 52, preferably F; and / or containing Y or V at position 55, preferably Y; - FR3-a contains or comprises the amino acid sequence of SEQ ID NO: 349, or an amino acid sequence that is at least 85%, 90%, or 95% identical to SEQ ID NO: 349, preferably containing T or K at position 92, preferably containing T, and / or containing D or G at position 93, preferably containing D; - FR4-a contains or consists of the amino acid sequence of SEQ ID NO: 350, or an amino acid sequence that is at least 85%, 90%, or 95% identical to SEQ ID NO: 350; V B This further includes one or more framework regions selected from the group consisting of FR1-b, FR2-b, FR3-b, and FR4-b, preferably all of the framework regions. - FR1-b contains or comprises the amino acid sequence of SEQ ID NO: 351 or SEQ ID NO: 352, or an amino acid sequence that is at least 85%, 90%, or 95% identical to SEQ ID NO: 351, preferably containing H or N at position 10, more preferably H, containing E, L, or K at position 11, preferably E, and / or containing R or H at position 22; - FR2-b contains or comprises the amino acid sequence of SEQ ID NO: 353, or an amino acid sequence that is at least 85%, 90%, or 95% identical to SEQ ID NO: 353, preferably containing R or K at position 43, more preferably R, containing E or Q at position 44, preferably E, containing M or P at position 46, more preferably P, and / or containing R or Q at position 48, more preferably Q; - FR3-b contains or comprises the amino acid sequence of SEQ ID NO: 354 or SEQ ID NO: 355, or an amino acid sequence that is at least 85%, 90%, or 95% identical to SEQ ID NO: 354, preferably containing D, A, E, R, K, Q, N, or S at position 84, more preferably containing D, A, E, Q, N, or S, more preferably containing D or A, and even more preferably containing D; - FR4-b contains or consists of the amino acid sequence of SEQ ID NO: 356, or an amino acid sequence that is at least 85%, 90%, or 95% identical to SEQ ID NO: 356.

[0227] The variants of antigen-binding proteins described herein are intended to be expressed as “at least 85% identical to the reference sequence” as defined in the “Definitions” section above, and are explicitly mentioned. For example, the amino acid sequences of FR1-a, FR2-a, FR3-a, FR4-a, FR1-b, FR2-b, FR3-b, and FR4-b may differ from the reference sequence SEQ ID NOs. 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, or 356 by at least one amino acid substitution, in particular by at least one conserved amino acid substitution and / or substitution with a canonical residue, as may be specified. A and V B The sequences FR1-a, FR2-a, FR3-a, and FR4-a, FR1-b, FR2-b, FR3-b, and FR4-b may differ from the reference sequence SEQ ID NO: 345, SEQ ID NO: 346, SEQ ID NO: 347, SEQ ID NO: 348, SEQ ID NO: 349, SEQ ID NO: 350, SEQ ID NO: 351, SEQ ID NO: 352, SEQ ID NO: 353, SEQ ID NO: 354, SEQ ID NO: 355, or SEQ ID NO: 356 by only conservative amino acid substitutions as necessary.

[0228] The amino acid sequence of the antigen-binding protein of the present invention and the corresponding DNA sequence can be modified and altered, respectively, and a functional antigen-binding protein or polypeptide with desirable properties can still be obtained. A and / or V B Modifications may be made (especially within the framework area or CDR).

[0229] V A and V B Preferably, compared to TCR R11P3D3, it contains an amino acid substitution at position 44 according to IMGT numbering. In embodiments where the antigen-binding protein is a TCR, these substitutions improve chain pairing (i.e., α-chain and β-chain pairing, or γ- and δ-chain pairing). A or V BIn this compound, the amino acid at position 44 may be substituted with an amino acid selected from the group consisting of Q, R, D, E, K, L, W, and V. Preferably, the amino acids present in SEQ ID NO: 347, SEQ ID NO: 348 (FR2-a), and SEQ ID NO: 353 (FR2-b), respectively, and which are amino acid pairs. VA 44K / VA V produces 44E A Substitution of W44K and V in the middle B The substitution in the middle is Q44E. Other suitable combinations are: VA 44Q / VB 44Q, VA 44D / VB 44R, VA 44R / VB 44D, VA 44E / VB 44K, VA 44D / VB 44K, VA 44K / VB 44D, VA 44R / VB 44E; VA 44E / VB 44R, VA 44L / VB 44W, VA 44W / VB 44L VA 44V / VB 44W, and VA 44W / VB It is 44V.

[0230] Additional substitutions and explanations can be found in U.S. Patent Application Publication No. 2018-0162922, which is incorporated herein by reference in its entirety.

[0231] Variable domain In one embodiment, V AThis includes or consists of the amino acid sequence of a TCR-derived variable domain contained in polypeptides of sequence numbers 100, 103, 105, 106, 111, 122, 124, 126, 128, 151, 155, 156, 157, 158, 159, 166, 167, 169, 171, 173, 175, 177, 178, 179, 180, 181, 183, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 285, 291, 295, 299, or 303, and V B These are sequence numbers 101, 102, 104, 107, 110, 119, 121, 131, 133, 143, 152, 153, 160, 161, 162, 163, 164, 165, 168, 170, 172, 174, 176, 182, 184, 185, 186, 216, 218, 220, 222, 224, 228, 230, 232, 234 , comprising or consisting of the amino acid sequence of a TCR-derived variable domain contained in polypeptides 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 282, 284, 296, or 300. A person skilled in the art can fully distinguish the amino acid sequence of the TCR-derived variable domain in the polypeptide chain of the above sequence numbers.

[0232] In one embodiment, - V AThis comprises or consists of the amino acid sequence of SEQ ID NO: 132, or an amino acid sequence that is at least 85%, 90%, or 95% identical to SEQ ID NO: 132, preferably comprising CDRa1 of SEQ ID NO: 16, CDRa2 of SEQ ID NO: 32, and CDRa3 of SEQ ID NO: 33, SEQ ID NO: 34, or SEQ ID NO: 9, and further may contain K or N at position 20, preferably K; L, M, or I at position 39, preferably L or I; K or W at position 44, preferably K; F or A at position 52, preferably F; Y or V at position 55, preferably Y; T or K at position 92, preferably T; and / or D or G at position 93, preferably D; - V B It contains or comprises the amino acid sequence of SEQ ID NO: 134, or an amino acid sequence that is at least 85%, 90%, or 95% identical to SEQ ID NO: 134, preferably containing CDRb1 of SEQ ID NO: 10, CDRb2 of SEQ ID NO: 36, and CDRb3 of SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 47, SEQ ID NO: 281, SEQ ID NO: 292, SEQ ID NO: 294, SEQ ID NO: 297, SEQ ID NO: 298, SEQ ID NO: 301, or SEQ ID NO: 283, and further may contain E, L, or K at position 11, preferably E, may contain R or H at position 22, may contain E or Q at position 44, preferably E, may contain P or M at position 46, preferably P, may contain Q or R at position 48, preferably Q, and / or may contain D, A, E, R, K, Q, N, or S at position 84, more preferably D, A, E, Q, N, or S, preferably D or A.

[0233] - V AIt comprises or consists of the amino acid sequence of SEQ ID NO: 132, or an amino acid sequence that is at least 85%, 90%, or 95% identical to SEQ ID NO: 132, and includes CDRa1 of SEQ ID NO: 16, CDRa2 of SEQ ID NO: 32, and CDRa3 of SEQ ID NO: 33, SEQ ID NO: 34, or SEQ ID NO: 9, and may further contain K or N at position 20, preferably K; L, M, or I at position 39, preferably L or I; K or W at position 44, preferably K; F or A at position 52, preferably F; Y or V at position 55, preferably Y; T or K at position 92, preferably T; and / or D or G at position 93, preferably D; - V B It contains or comprises the amino acid sequence of SEQ ID NO: 134, or an amino acid sequence that is at least 85%, 90%, or 95% identical to SEQ ID NO: 134, and includes CDRb1 of SEQ ID NO: 10, CDRb2 of SEQ ID NO: 36, and CDRb3 of SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 47, SEQ ID NO: 281, SEQ ID NO: 292, SEQ ID NO: 294, SEQ ID NO: 297, SEQ ID NO: 298, SEQ ID NO: 301, or SEQ ID NO: 283, and further may contain E, L, or K at position 11, preferably E, may contain R or H at position 22, may contain E or Q at position 44, preferably E, may contain P or M at position 46, preferably P, may contain Q or R at position 48, preferably Q, and / or may contain D, A, E, R, K, Q, N, or S at position 84, more preferably D, A, E, Q, N, or S, preferably D or A. It is preferable.

[0234] In a preferred embodiment, - V A This includes or consists of the amino acid sequence of SEQ ID NO: 132, SEQ ID NO: 129, SEQ ID NO: 137, or SEQ ID NO: 142. - V B This includes or consists of the amino acid sequence of SEQ ID NO: 134, SEQ ID NO: 130, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, or SEQ ID NO: 148.

[0235] - V A It contains or consists of the amino acid sequence of SEQ ID NO: 132, and V B This includes or consists of the amino acid sequence of sequence number 134; - V A It contains or consists of the amino acid sequence of SEQ ID NO: 132, and V B This includes or consists of the amino acid sequence of SEQ ID NO: 135; - V A It contains or consists of the amino acid sequence of SEQ ID NO: 132, and V B This includes or consists of the amino acid sequence of SEQ ID NO: 140; - V A It contains or consists of the amino acid sequence of SEQ ID NO: 132, and V B This includes or consists of the amino acid sequence of sequence number 136; - V A It contains or consists of the amino acid sequence of sequence number 137, and V B This includes or consists of the amino acid sequence of sequence number 134; - V A It contains or consists of the amino acid sequence of sequence number 137, and V B is either containing or consisting of the amino acid sequence of SEQ ID NO: 135; or - V A It contains or consists of the amino acid sequence of sequence number 137, and V B This includes or consists of the amino acid sequence of SEQ ID NO: 134. This is particularly preferable.

[0236] Most preferably, V AThis contains or consists of the amino acid sequence of sequence number 132, V B This includes or consists of the amino acid sequence of SEQ ID NO: 134, 135, or 140 (especially SEQ ID NO: 135). Therefore, V A may contain or consist of the amino acid sequence of sequence number 132, V B may contain or consist of the amino acid sequence of SEQ ID NO: 135. Alternatively, V A may contain or consist of the amino acid sequence of sequence number 132, V B This may include or consist of the amino acid sequence of SEQ ID NO: 140.

[0237] The variants of antigen-binding proteins described herein are intended and explicitly referred to using the expression “at least 85% identical to the reference sequence” as defined in the above definition section herein. For example, V A and V B The sequences may differ from the reference sequences of SEQ ID NOs. 132 and 134, respectively, by at least one amino acid substitution, in particular by at least one conservative amino acid substitution and / or substitution with a canonical residue. In particular, V A and V B The sequences may differ from the reference sequences of SEQ ID NO: 132 and SEQ ID NO: 134, respectively, solely through conservative amino acid substitutions.

[0238] The amino acid sequence of the antigen-binding protein of the present invention and the corresponding DNA sequence can be modified and altered, and a functional antigen-binding protein or polypeptide with desirable properties can still be obtained.

[0239] In one embodiment, the antigen-binding protein of the present invention is as follows: (i) One or more additional antigen-binding sites: (ii) A transmembrane region which may include a cytoplasmic signaling region; (iii) Diagnostic agents; (iv) Therapeutic drugs; or (v) Modified parts of PK This further includes one or more of the following.

[0240] If components (i) to (v) listed above are polypeptides fused to the antigen-binding protein of the present invention, this antigen-binding protein may also be referred to as a "TCR fusion protein."

[0241] Further antigen-binding sites are preferably derived from antibodies.

[0242] The "transmembrane region" related to the present invention may be, for example, a TCR alpha or beta transmembrane domain.

[0243] The "cytoplasmic signaling region" may be, for example, an intracellular domain of TCR alpha or beta.

[0244] "Diagnostic agent" as used herein means a detectable molecule or substance (e.g., a fluorescent molecule, a radioactive molecule, or any other label known in the art to produce a signal (directly or indirectly)).

[0245] Known "fluorescent molecules" in the art include fluorescein isothiocyanate (FITC), phycoerythrin (PE), fluorophores used in blue lasers (e.g., PerCP, PE-Cy7, PE-Cy5, FL3, and APC or Cy5, FL4), and fluorophores used in red, violet, or UV lasers (e.g., Pacific blue, Pacific orange).

[0246] As a "radioactive molecule", I 123 , I 124 In 111 Re 186 Re 188 , Tc 99Examples of radioactive atoms for scintigraphy studies include, but are not limited to, those listed above. The antigen-binding proteins of the present invention may also include spin labels for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, or MRI) (e.g., iodine-123, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron).

[0247] Such diagnostic agents may be directly bound (i.e., physically linked) to antigen-binding proteins, or they may be linked indirectly.

[0248] In this specification, "therapeutic agent" refers to a drug that has a therapeutic effect. The terms therapeutic agent and therapeutic drug are used interchangeably herein. In one embodiment, the therapeutic agent may be a cytotoxic agent or a growth inhibitor such as a radioisotope.

[0249] "Growth inhibitors" or "antiproliferative agents" (these terms may be used interchangeably) refer to compounds or compositions that inhibit the growth of cells (particularly tumor cells) either in vitro or in vivo.

[0250] The term “cytotoxic agent,” as used herein, refers to a substance that inhibits or prevents the function of a cell and / or causes cell destruction. The term “cytotoxic agent” is intended to include chemotherapeutic agents, enzymes, antibiotics, and toxins (e.g., low molecular weight toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin) (including fragments and / or variants thereof), as well as various antitumor or anticancer agents disclosed below. In some embodiments, cytotoxic formulations include taxoids, vinca, taxanes, maytansinoids or maytansinoid analogs (e.g., DM1 or DM4), small drugs, tomaimycin or pyrrolobenzodiazepine derivatives, cryptophycin derivatives, leptomycin derivatives, auristatin or drastatin analogs, prodrugs, topoisomerase II inhibitors, DNA alkylating agents, antitubulin agents, CC-1065, or CC-1065 analogs.

[0251] The term "radioisotope" is intended to include radioisotopes suitable for cancer treatment, for example, At 211 , Bi 212 Er 169 , I 131 , I 125 , Y 90 In 111 , P 32 Re 186 Re 188 Sm 153 Sr 89 It is intended to include, and radioactive isotopes of Lu. Such radioactive isotopes generally emit primarily beta rays. In one embodiment, the radioactive isotope is an alpha-emitting isotope, more precisely, thorium-227 which emits alpha rays.

[0252] In some embodiments, the antigen-binding protein of the present invention is covalently bound to at least one growth inhibitor, either directly or via a cleavable or non-cleavable linker. The antigen-binding protein to which such at least one growth inhibitor is bound may also be referred to as a conjugate. The cleavable linker facilitates the release of the cytotoxic agent or growth inhibitor from the antigen-binding protein within the cell. For example, an acid-unstable linker, a peptidase-sensitive linker, an esterase-unstable linker, a photo-unstable linker, or a disulfide-containing linker may be used (see, for example, U.S. Patent No. 5,208,020). The linker may also be a “non-cleavable linker” (e.g., an SMCC linker), which may, in some cases, provide better resistance.

[0253] The preparation of such conjugates (e.g., immunoconjugates) is described in International Publication No. 2004 / 091668, or in Hudecz, F., Methods Mol. Biol. 298: 209-223 (2005) and Kirin et al., Inorg Chem. 44(15): 5405-5415 (2005) (the contents of which are incorporated herein by reference in their entirety), and those skilled in the art may adapt these to the preparation of the antigen-binding proteins of the present invention to which at least one such growth inhibitor is conjugated.

[0254] Alternatively, a fusion protein comprising the antigen-binding protein of the present invention and a cytotoxic polypeptide or a proliferation-inhibiting polypeptide can be prepared by recombinant technology or peptide synthesis. The length of the DNA may include each region encoding two adjacent parts of the conjugate, or it may include each region encoding two parts of the conjugate separated by a region encoding a linker peptide that does not disrupt the desired properties of the conjugate.

[0255] The antigen-binding protein of the present invention can also be used in enzyme-dependent prodrug therapy by conjugating a polypeptide to a prodrug-activating enzyme that converts a prodrug (e.g., a peptidyl chemotherapeutic agent; see International Publication No. 81 / 01145) into an active anticancer agent (e.g., see International Publication No. 88 / 07378 and U.S. Patent No. 4,975,278).

[0256] In this specification, the "PK modification portion" refers to a site that modifies the pharmacokinetics of the antigen-binding protein of the present invention. Accordingly, this portion modifies, in particular, the in vivo half-life and distribution of the antigen-binding protein of the present invention. In preferred embodiments, the PK modification portion increases the half-life of the antigen-binding protein. Examples of PK modification include, but are not limited to, the following: PEG [Dozier et al., (2015) Int J Mol Sci. Oct 28;16(10):25831-64, and Jevsevar et al., (2010) Biotechnol J.Jan;5(1):113-28], PAS modification [Schlapschy et al., (2013) Protein Eng Des Sel. Aug;26(8):489-501], albumin [Dennis et al., (2002) J Biol Chem. Sep 20;277(38):35035-43)], antibodies and / or unstructured polypeptides. c Part [Schellenberger et al., (2009) Nat Biotechnol. Dec; 27(12):1186-90].

[0257] In one embodiment, the antigen-binding protein of the present invention further comprises one or more of the following: an enzyme, a cytokine (e.g., human IL-2, IL-7, or IL-15), a nanocarrier, or a nucleic acid.

[0258] Second antigen-binding site In a preferred embodiment, the antigen-binding protein has an antibody light chain variable domain (V L) and antibody heavy chain variable domain (V H ) further includes. Variable Domain V L and variable domain V H These two elements together form an antigen-binding site. From this point forward, this antigen-binding site may also be referred to as the "second antigen-binding site." L and V H The antigen-binding site formed by this process preferably binds to the antigen of the effector cell and recruits the effector cell to the tumor, and can therefore also be called a "recruiter." In relation to the present invention, "effector cell" refers to a T cell or a natural killer cell (NK cell).

[0259] In a preferred embodiment, V H and V H CD2, CD3 (e.g., CD3γ, CD3δ, and CD3ε chains), CD4, CD5, CD7, CD8, CD10, CD11b, CD11c, CD14, CD16, CD18, CD22, CD25, CD28, CD32a, CD32b, CD33, CD41, CD41b, CD42a, CD42b, CD44, CD45RA, CD49, CD55, CD56, CD61, CD64, CD68, CD90, CD94, CD95, CD117, CD123, CD125, CD134, CD137, CD152, CD163, CD193, CD203c, CD235a, CD278, CD279, CD287, Nkp46, NKG2D, GITR, F c It binds to antigens selected from the group consisting of εRI, TCRα / β, TCRγ / δ, HLA-DR, and 4-1 BB, or combinations thereof, and / or V H and V L It binds to effector cells. "This combination" refers to two or more complexes of the aforementioned antigens (e.g., the TCRα / β CD3 complex). Preferably, the antigens are CD3, the TCRα / β CD3 complex, or CD28, and more preferably CD3 or the TCRα / β CD3 complex.

[0260] In the case of targeting the TCR-CD3 complex, V derived from the CD3-specific humanized antibody hUCHT1 (Zhu et al., Identification of heavy chain residues in a humanized anti-CD3 antibody important for efficient antigen binding and T cell activation. J Immunol, 1995, 155, 1903-1910) H and V L The domain may be used, in particular V derived from UCHT1 variants UCHT1-V17, UCHT1-V17opt, UCHT1-V21, or UCHT1-V23. H and V L Domains may be used, preferably V derived from UCHT1-V17. H and V L A domain may be used, more preferably V including or consisting of sequence number 109. H V including or consisting of sequence number 108 L Alternatively, V derived from the antibody BMA031, which targets the TCRα / β CD3 complex, and its humanized version (Shearman et al., Construction, expression and characterization of humanized antibodies directed against the human alpha / beta T cell receptor, J Immunol, 1991, 147, 4366-73) may be used. H and V L The domain may be used, in particular V derived from the BMA031 variant BMA031(V36) or BMA031(V10). H and V L Domains may be used, preferably V derived from BMA031(V36). H and V LA domain may be used, and V may include or consist of sequence numbers 112, or sequence numbers 114 (A02), or sequence number 115 (D01), or sequence number 116 (A02_H90Y), or sequence number 117 (D01_H90Y). H V including or consisting of sequence number 113 L This can be used. Another option is V derived from CD3ε-specific antibody H2C (described in European Patent No. 2155783). H and V L Domains may be used, in particular V which includes or consists of sequence numbers 118, or sequence numbers 123 (N100D), or sequence numbers 125 (N100E), or sequence number 127 (S101A). H V including or consisting of sequence number 120 L It may be used. All location and CDR definitions follow the Kabat numbering scheme.

[0261] In some embodiments, V H and V L Both bind to the TCRα / β CD3 complex, and V H teeth, - Heavy chain complementarity determination region 1 (HCDR1) containing the amino acid sequence of SEQ ID NO: 381 (SYVMH), - HCDR2 containing the amino acid sequence YINPYNDVTKYX1X2KFX3G (SEQ ID NO: 382) (wherein X1 is A or N; X2 is E or Q; and / or X3 is Q or K), - HCDR3, and - Heavy Chain Framework Region (HFR) 1-4 Includes, V L teeth, - Light chain complementarity determination region 1 (LCDR1) containing the amino acid sequence of sequence number 383 (SATSSVSYMH) - LCDR2 containing the amino acid sequence of SEQ ID NO: 384 (DTSKLAS), and - LCDR3 Includes, - At least one amino acid of HCDR1 containing the amino acid sequence of SEQ ID NO: 1 which is not positively charged, and / or at least one amine of HCDR2 containing the amino acid sequence of SEQ ID NO: 2, are substituted with a positively charged amino acid; and / or - At least one amino acid of LCDR1 containing the amino acid sequence of SEQ ID NO: 3 which is not positively charged, and / or at least one amine of LCDR2 containing the amino acid sequence of SEQ ID NO: 4, is substituted with a positively charged amino acid; and / or - HFR3 contains a tyrosine (Y) residue at position 90 according to Kabat numbering.

[0262] Preferably, the antigen-bound polypeptide is In heavy chains, - The positively charged amino acid at position 31 is R, K, or H; - The positively charged amino acid at position 53 is R, K, or H; and / or - The positively charged amino acid at position 54 is R or K; and / or In light chains, - The positively charged amino acid at position 31 is R or K; and / or - The positively charged amino acid at position 56 is either R or K.

[0263] In some embodiments, V H This includes sequences selected from the group consisting of sequence numbers 112, 114-117, and 366-376, V L This includes sequences selected from the group consisting of sequence numbers 113 and 377-380. Preferably, V H This includes or consists of the amino acid sequence of sequence number 112, or 114-117, V L This includes or consists of the amino acid sequence of SEQ ID NO: 113 or 378 (preferably 113).

[0264] CD28 is expressed on T cells and can generate costimulatory signals necessary for T cell activation. CD28 plays a crucial role in T cell proliferation and survival, cytokine production, and the development of T helper type 2.

[0265] "CD134" is also known as Ox40. CD134 / OX40 is expressed 24–72 hours after activation and can be used to define secondary costimulatory molecules.

[0266] "4-1BB" can bind to the 4-1 BB ligand on antigen-presenting cells (APCs), thereby generating a co-stimulatory signal to T cells.

[0267] "CD5" is another example of a receptor primarily found on T cells, although CD5 is also found on B cells at low levels.

[0268] CD95 is another example of a receptor that modifies T cell function and is also known as a Fac receptor that mediates apoptotic signaling mediated by Fas ligand expressed on the surface of other cells. CD95 has been reported to modulate the TCR / CD3-driven signaling pathway in quiescent T lymphocytes.

[0269] Examples of "NK cell-specific receptor molecules" include CD16, low-affinity Fc receptors, and NKG2D.

[0270] Examples of receptor molecules present on the surface of both T cells and natural killer (NK) cells include CD2 and further members of the CD2 superfamily. CD2 can function as a costimulatory molecule on both T cells and NK cells.

[0271] Bispecific antigen-binding proteins and multi-characteristic antigen-binding proteins Therefore, the antigen-binding protein of the present invention preferably forms a first antigen-binding site specific to the PRAME-004:MHC complex. A and V B, and V which forms a second antigen-binding site that can bind to effector cells (preferably T cells) L and V H Includes. V A , V B , V L , and V H It may be located on a single polypeptide chain or on several polypeptide chains (preferably two polypeptide chains). A , V B , V L , and V H In addition, the antigen-binding protein of the present invention may or may not contain a dimerization domain (preferably a constant-state immunoglobulin domain).

[0272] In some embodiments, V A , V B , V L , and V H It is located on two polypeptide chains. Preferably, each polypeptide chain contains two variable domains. One polypeptide chain is V A It includes, and the other polypeptide chain is V B Preferably, it contains V A Polypeptide chains containing V L and V H Including one of the following, V B Polypeptide chains containing V L and V H Includes the other of the two. A Polypeptide chains containing V L and V H Both include V B Polypeptide chains containing V L V H It is also possible that it does not include, and vice versa. Another possibility is that one polypeptide chain is V A It contains one polypeptide chain V B It includes and the third polypeptide chain is V L and V H It includes.

[0273] In a preferred embodiment, the antigen-binding protein comprises first and second polypeptide chains. The first polypeptide chain is given by formula [Ia]: V1-L1-D1-L2-V2-L3-D2[Ia] It is represented by, The second polypeptide chain is given by formula [IIa] V3-L4-D3-L5-V4-L6-D4[IIa] It is represented by, During the ceremony, - V1, V2, V3, and V4 are variable domains, and one is V A And one of V1 to V4 is V B And one is V L And one of them is V H and; - D1, D2, D3, and D4 are dimerization domains and may or may not be present, D1 and D3, and D2 and D4 bind specifically to each other, and at least one pair of D1 and D3 or D2 and D4 is present. - L1, L2, L3, L4, L5, and L6 are linkers; L1 and L4 are present, and L2, L3, L5, and L6 may or may not be present.

[0274] One of V1 and V2 is V A And one of V3 and V4 is V B And of the remaining two variable domains, one is V L The other is V H It is preferable that V A and V B It is located on different polypeptide chains, V L and V H These are located on different polypeptide chains.

[0275] The dimerization domain is preferably a heterodimerization domain that mediates heterodimerization between a first polypeptide chain and a second polypeptide chain, but does not mediate homodimerization between two first polypeptide chains or two second polypeptide chains. In a preferred embodiment, a pair of dimerization domains (e.g., D1 and D3, and / or D2 and D4) are immunoglobulin constant domains, e.g., antibody-derived C L and C H1 , or C L -F c and C H1 -F c , or C derived from TCR α and C β , or a pair of C H3 Domain, or a pair of F c Including the domain, C H3 Domain and F c The domain preferably contains introduced mutations that force heterodimerization, such as knob-into-hole mutations.

[0276] In a more preferred embodiment, the antigen-binding protein comprises first and second polypeptide chains, The first polypeptide chain is given by formula [Ib]: V1-L1-V2-L3-D2[Ib] It is represented by, The second polypeptide chain is given by formula [IIb]: V3-L4-V4-L6-D4[IIb] It is represented by, During the ceremony, - V1, V2, V3, V4 are variable domains, and one is V A And one is, V B And one is, V L And one is, V H and; - D2 and D4 are dimerization domains that specifically bind to each other, preferably Fc domains; - L1, L3, L4, and L6 are linkers, and L3 and L6 may or may not be present.

[0277] As explained with respect to equations Ia and IIA, V A and V B It is located on different polypeptide chains, V L and V H Preferably, the dimerization domain is located on different polypeptide chains, and the dimerization domain is a heterodimerization domain.

[0278] In a preferred embodiment, D2 and D4 are a pair of F c Domain F c1 and F c2 And in particular, D2 is F c1 And D4 is F c2 And here, F c1 and F c2 These are identical or different, preferably different, and preferably include mutations that force heterodimerization. In one embodiment, F c1 This contains or consists of the amino acid sequence (whole) of sequence number 150, F c2 This includes or consists of the amino acid sequence (nob) of sequence number 149, and vice versa. In particular, F c1 However, V L It is located on a polypeptide chain containing F c2 However, V H If located on a polypeptide chain containing F c1 It contains or consists of the amino acid sequence (nob) of sequence number 149, F c2 This contains or consists of the amino acid sequence (whole) of sequence number 150, F c1 However, V H It is located on a polypeptide chain containing F c2 However, V L If located on a polypeptide chain containing F c1 This contains or consists of the amino acid sequence (whole) of sequence number 150, F c2 This includes or consists of the amino acid sequence (nob) of sequence number 149.

[0279] In antigen-binding proteins containing first and second polypeptide chains represented by formulas Ia and IIa or Ib and IIb, V A and V B , and V L and V H Those skilled in the art will understand that the orientation may be parallel, as in the DVD format, or cross-oriented, as in the CODV format.

[0280] In formulas Ia and IIa, or Ib and IIb, V A , V B , V L , and V H It may have the following orientations: (1) V1 is V H Therefore, V2 is V B Therefore, V3 is V A And V4 is V L Is it; (2) V1 is V B Therefore, V2 is V H Therefore, V3 is V L And V4 is V A Is it; (3) V1 is V B Therefore, V2 is V L Therefore, V3 is V H And V4 is V A Is it; (4) V1 is V L Therefore, V2 is V B Therefore, V3 is V A And V4 is V H Is it; (5) V1 is V H Therefore, V2 is V B Therefore, V3 is V L And V4 is V A Is it; (6) V1 is V B Therefore, V2 is V H Therefore, V3 is V A And V4 is V L Is it; (7) V1 is VL Therefore, V2 is V B Therefore, V3 is V H And V4 is V A Is it; (8) V1 is V B Therefore, V2 is V L Therefore, V3 is V A And V4 is V H Is it; (9) V1 is V H Therefore, V2 is V L Therefore, V3 is V A And V4 is V B Is it; (10) V1 is V L Therefore, V2 is V H Therefore, V3 is V A And V4 is V B Is it; (11) V1 is V H Therefore, V2 is V L Therefore, V3 is V B And V4 is V A is; or (12) V1 is V L Therefore, V2 is V H Therefore, V3 is V B And V4 is V A That is the case.

[0281] V A , V B , V L , and V H It is preferable that it has the orientation described in (1) to (8), that is, V A and V B It is located on different polypeptide chains, V L and V H Preferably, V is located on different polypeptide chains. A , V B , V L , and V H It has the orientation described in (1) to (4), that is, V A and V B, and V L and V H It has cross-orientation.

[0282] Linkers L1, L2, L3, L4, and L5 are defined in the “Definitions” section above in this specification. In some embodiments, certain linker lengths may be preferred in certain formats. However, knowledge of linker lengths and amino acid sequences is part of the general knowledge of the art, and linkers and linker amino acid sequences for various formats are part of the art and are disclosed in the disclosures cited above.

[0283] The antigen-binding protein of the present invention is particularly preferably in TCER® format. In embodiments of the TCER® format, the antigen-binding protein comprises a first polypeptide chain and a second polypeptide chain represented by the formulas [IIa] and [IIb] defined above, where, - V1 is V H Therefore, V2 is V B Therefore, V3 is V A And V4 is V L Is it; V1 is V B Therefore, V2 is V H Therefore, V3 is V L And V4 is V A Is it; V1 is V B Therefore, V2 is V L Therefore, V3 is V H And V4 is V A is; or V1 is V L Therefore, V2 is V B Therefore, V3 is V A And V4 is V H and; - L3 and L6 do not exist; - L1 and L4 preferably contain or consist of the amino acid sequence of SEQ ID NO: 214; - D2 and D4 are a pair of F c Domain F c1 and F c2 And F c1 and F c2 These include mutations that are different and force heterodimerization (preferably "knob-into-hole" mutations).

[0284] In a preferred embodiment, - V L It contains or consists of the amino acids of sequence number 108, and V H It contains or consists of the amino acids of sequence number 109, or - V L It contains or consists of the amino acids of sequence number 113, and V H It contains or consists of the amino acids of SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, or SEQ ID NO: 117, or - V L It contains or consists of the amino acids of sequence number 120, and V H It contains or consists of the amino acids of SEQ ID NO: 118, SEQ ID NO: 123, SEQ ID NO: 125, or SEQ ID NO: 127.

[0285] In a particularly preferred embodiment, the antigen-binding protein comprises a first polypeptide chain selected from SEQ ID NOs: 100, 103, 105, 106, 111, 122, 126, 128, 151, 155, 156, 157, 158, 159, 166, 167, 169, 171, 173, 175, 177, 178, 179, 180, 181, 183, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 285, 291, 295, 299, and 303, and SEQ ID NOs: 101, 102, 104, 107 , comprising a second polypeptide chain selected from 110, 119, 121, 131, 133, 143, 152, 160, 161, 162, 163, 164, 165, 168, 170, 172, 174, 176, 182, 184, 185, 186, 216, 218, 220, 222, 224, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 282, 284, 296, or 300.

[0286] In a more preferred embodiment, the antigen-binding protein is selected from SEQ ID NOs: 100, 103, 105, 151, 156, 158, 166, 167, 175, 178, 180, 183, 193, 285, 291, 295, 299, and 303, and more preferably selected from SEQ ID NOs: 100, 103, 105, 167, 183, 193, 285, 291, 295, 299, and 303. The molecule comprises a first polypeptide chain and a second polypeptide chain selected from SEQ ID NOs: 101, 102, 104, 160, 161, 162, 163, 164, 165, 170, 172, 174, 176, 182, 185, 186, 284, 296, or 300, and more preferably selected from SEQ ID NOs: 101, 102, 104, 160, 162, 176, 186, 284, 296, or 300.

[0287] In the most preferred embodiment, the antigen-binding protein includes: - The first polypeptide chain of SEQ ID NO: 100, and the second polypeptide chain of SEQ ID NO: 101, or - The first polypeptide chain of SEQ ID NO: 103, and the second polypeptide chain of SEQ ID NO: 102, or - The first polypeptide chain of SEQ ID NO: 105, and the second polypeptide chain of SEQ ID NO: 104, or - The first polypeptide chain of SEQ ID NO: 167, and the second polypeptide chain of SEQ ID NO: 160, or - The first polypeptide chain of SEQ ID NO: 183, and the second polypeptide chain of SEQ ID NO: 176, or - The first polypeptide chain of SEQ ID NO: 193, and the second polypeptide chain of SEQ ID NO: 186, or - The first polypeptide chain of SEQ ID NO: 285, and the second polypeptide chain of SEQ ID NO: 284, or - The first polypeptide chain of SEQ ID NO: 291, and the second polypeptide chain of SEQ ID NO: 284, or - The first polypeptide chain of SEQ ID NO: 295, and the second polypeptide chain of SEQ ID NO: 186, or - The first polypeptide chain of SEQ ID NO: 295, and the second polypeptide chain of SEQ ID NO: 296, or - The first polypeptide chain of SEQ ID NO: 299, and the second polypeptide chain of SEQ ID NO: 162, or - The first polypeptide chain of SEQ ID NO: 285, and the second polypeptide chain of SEQ ID NO: 300, or - The first polypeptide chain of SEQ ID NO: 303, and the second polypeptide chain of SEQ ID NO: 162, or - The first polypeptide chain of SEQ ID NO: 291, and the second polypeptide chain of SEQ ID NO: 300, or - The first polypeptide chain of SEQ ID NO: 151, and the second polypeptide chain of SEQ ID NO: 284, or - The first polypeptide chain of SEQ ID NO: 156, and the second polypeptide chain of SEQ ID NO: 162, or - The first polypeptide chain of SEQ ID NO: 158, and the second polypeptide chain of SEQ ID NO: 284, or - The first polypeptide chain of SEQ ID NO: 158, and the second polypeptide chain of SEQ ID NO: 300, or - The first polypeptide chain of SEQ ID NO: 303, and the second polypeptide chain of SEQ ID NO: 161, or - The first polypeptide chain of SEQ ID NO: 303, and the second polypeptide chain of SEQ ID NO: 163, or - The first polypeptide chain of SEQ ID NO: 291, and the second polypeptide chain of SEQ ID NO: 164, or - The first polypeptide chain of SEQ ID NO: 291, and the second polypeptide chain of SEQ ID NO: 170, or - The first polypeptide chain of SEQ ID NO: 291, and the second polypeptide chain of SEQ ID NO: 172, or - The first polypeptide chain of SEQ ID NO: 291, and the second polypeptide chain of SEQ ID NO: 174, or - The first polypeptide chain of SEQ ID NO: 166, and the second polypeptide chain of SEQ ID NO: 170, or - The first polypeptide chain of SEQ ID NO: 166, and the second polypeptide chain of SEQ ID NO: 172, or - The first polypeptide chain of SEQ ID NO: 166, and the second polypeptide chain of SEQ ID NO: 174, or - The first polypeptide chain of SEQ ID NO: 291, and the second polypeptide chain of SEQ ID NO: 182, or - The first polypeptide chain of SEQ ID NO: 291, and the second polypeptide chain of SEQ ID NO: 185, or - The first polypeptide chain of SEQ ID NO: 175, and the second polypeptide chain of SEQ ID NO: 186, or - The first polypeptide chain of SEQ ID NO: 178, and the second polypeptide chain of SEQ ID NO: 186, or - The first polypeptide chain of SEQ ID NO: 180, and the second polypeptide chain of SEQ ID NO: 186, especially, - The first polypeptide chain of SEQ ID NO: 100, and the second polypeptide chain of SEQ ID NO: 101, or - The first polypeptide chain of SEQ ID NO: 103, and the second polypeptide chain of SEQ ID NO: 102, or - The first polypeptide chain of SEQ ID NO: 105, and the second polypeptide chain of SEQ ID NO: 104, or - The first polypeptide chain of SEQ ID NO: 158, and the second polypeptide chain of SEQ ID NO: 300, or - The first polypeptide chain of SEQ ID NO: 167, and the second polypeptide chain of SEQ ID NO: 160, or - The first polypeptide chain of SEQ ID NO: 183, and the second polypeptide chain of SEQ ID NO: 176, or - The first polypeptide chain of SEQ ID NO: 193, and the second polypeptide chain of SEQ ID NO: 186, or - The first polypeptide chain of SEQ ID NO: 285, and the second polypeptide chain of SEQ ID NO: 284, or - The first polypeptide chain of SEQ ID NO: 291, and the second polypeptide chain of SEQ ID NO: 164, or - The first polypeptide chain of SEQ ID NO: 291, and the second polypeptide chain of SEQ ID NO: 284, or - The first polypeptide chain of SEQ ID NO: 295, and the second polypeptide chain of SEQ ID NO: 186, or - The first polypeptide chain of SEQ ID NO: 295, and the second polypeptide chain of SEQ ID NO: 296, or - The first polypeptide chain of SEQ ID NO: 299, and the second polypeptide chain of SEQ ID NO: 162, or - The first polypeptide chain of SEQ ID NO: 285, and the second polypeptide chain of SEQ ID NO: 300, or - The first polypeptide chain of SEQ ID NO: 303, and the second polypeptide chain of SEQ ID NO: 162, or - The first polypeptide chain of SEQ ID NO: 291, and the second polypeptide chain of SEQ ID NO: 300, Furthermore, especially, - The first polypeptide chain of SEQ ID NO: 158, and the second polypeptide chain of SEQ ID NO: 300, or - The first polypeptide chain of SEQ ID NO: 291, and the second polypeptide chain of SEQ ID NO: 164.

[0288] Therefore, in the most preferred embodiment, the antigen-binding protein may include a first polypeptide chain of SEQ ID NO: 158 and a second polypeptide chain of SEQ ID NO: 300.

[0289] scTCR In some embodiments, the first and second polypeptides, therefore V A and V B It is located on a single polypeptide chain. In such embodiments, the antigen-binding protein of the present invention may be described as a single-chain TCR. However, depending on the FR sequence and constant domain contained in the antigen-binding protein, it may also be referred to as a single-chain antibody or a single-chain TCR antibody molecule, as described above.

[0290] An scTCR may comprise a variable domain derived from or containing at least a CDR derived from a first TCR, a variable domain derived from or containing at least a CDR derived from a second TCR, and a constant domain of the first or second TCR. In other words, a single-stranded TCR comprises a variable domain derived from one TCR (e.g., an α or γ chain) and an entire chain of another TCR (e.g., a β or δ chain), or vice versa. Furthermore, an scTCR may comprise one or more linkers (preferably peptide linkers) linking the domains together. Such scTCRs of the present invention are also provided, which are fused to human cytokines (e.g., IL-2, IL-7, or IL-15).

[0291] In one embodiment, the single-stranded TCR is V A -L t -V B , V B -L t -V A , V A -C α -L t -V B , V A-C β -L t -V B , V A -L t -V B -C β , V A -L t -V B -C α , V A -C α -L t -V B -C β , V A -C b -L t -V B -C α (preferably, V A -L t -V B , V B -L t -V A It exists as one of the single-stranded formats selected from the group consisting of ), where V A is the first variable domain as defined above in this specification, and V B This is the second variable domain as defined above in this specification, C α and C β These are the TCR alpha constant domain and the TCR beta constant domain, respectively, which are present or absent. t This is a linker, which may or may not exist, as defined in the above definition section of this specification.

[0292] In certain embodiments, the antigen-binding protein of the present invention comprises an amino acid sequence from any of SEQ ID NOs. 79-87 or 89-92, or an amino acid sequence that is at least 85% identical to SEQ ID NOs. 79-87 or 89-92, and preferably an scTCR comprising the amino acid sequence of SEQ ID NO. 87.

[0293] scTCR-Fab A single-stranded TCR has a further variable domain ligated to either the C-terminus or N-terminus, in particular the V described above. L and / or VH It may include.

[0294] In one embodiment, such further variable domains are linked to the linker L k They can be connected via a linker L. In one preferred embodiment, linker L k This refers to either a linker as defined above in this specification, or the hinge-C of the amino acid sequence of SEQ ID NO: 360. H1 It is an array.

[0295] In a particular embodiment, the antigen-binding protein of the present invention is V A , V B , and V L or V H (preferably, V H A first polypeptide chain containing ) and V L and V H The other (preferably, V L The scTCR-Fab comprises a first polypeptide chain comprising or consisting of the amino acid sequence of any of SEQ ID NOs. 94-98, or an amino acid sequence identical to or at least 85% of SEQ ID NOs. 94-98, and a second polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NOs. 93, or an amino acid sequence identical to or at least 85% of SEQ ID NOs. 93.

[0296] full length TCR In another embodiment, the antigen-binding protein of the present invention comprises two polypeptide chains, V A It is contained in the (full-length) TCRα or γ chain; V B The (full-length) TCR is contained in a β or δ chain. In such embodiments, the antigen-binding protein preferably has the structure of the conventional αβ TCR or γδ TCR described above. In one embodiment, the TCR is an αβ TCR and comprises an α-chain constant domain (TRAC) sequence according to SEQ ID NO: 361 and a β-chain constant domain (TRBC1 or TRBC2) sequence according to SEQ ID NO: 362.

[0297] In one embodiment, the TCR constant domain sequence may be derived from any suitable species (e.g., any mammal, e.g., human, rat, monkey, rabbit, donkey, or mouse, preferably human). In some preferred embodiments, the TCR constant domain sequence may be slightly modified, for example, by introducing a heterologous sequence (preferably a mouse sequence) that can increase TCR expression and stability. Similarly, further stabilizing mutations known in the art (e.g., International Publication No. 2018 / 104407, PCT / EP2018 / 069151, International Publication No. 2011 / 044186, International Publication No. 2014 / 018863, e.g., substitution of undesirable amino acids in the variable region, and / or introduction of disulfide crosslinks between TCR C domains, and removal of unpaired cysteine) may be introduced.

[0298] In particular, the TCR constant domain sequence may be modified by cleavage or substitution to delete the native disulfide bond between Cys4 in exon 2 of TRAC and Cys2 in exon 2 of TRBC1 or TRBC2. The constant domain sequences of the alpha and / or delta chains may also be modified by substitution of cysteine ​​residues at Thr48 of TRAC and Ser57 of TRBC1 or TRBC2, where the cysteine ​​forms a disulfide bond between the alpha and beta constant domains of the TCR. TRBC1 or TRBC2 may further include a cysteine-to-alanine mutation at position 75 of the constant domain and an asparagine-to-aspartic acid mutation at position 89 of the constant domain. The constant domain may further or / or include further mutations, substitutions, or deletions of the native TRAC and / or TRBC1 / 2 sequences. The terms TRAC and TRBC1 / 2 encompass naturally occurring polymorphic variants (e.g., N to K at position 4 of TRAC) (Bragado et al Int Immunol. 1994 Feb;6(2):223-30).

[0299] The present invention also includes particles that display antigen-binding proteins (in particular TCRs), and the inclusion of such particles in a library of particles. Examples of such particles include, but are not limited to, phages, yeast, ribosomes, or mammalian cells. Methods for producing such particles and libraries are known in the art (see, for example, International Publication No. 2004 / 044004; International Publication No. 01 / 48145, Chervin et al. (2008) J. Immuno. Methods 339.2: 175-184).

[0300] Nucleic acids, vectors, and recombinant host cells In a second aspect, the present invention relates to an isolated nucleic acid comprising or comprising a sequence encoding an antigen-binding protein according to the first aspect of the present invention.

[0301] In relation to the present invention, the term "nucleic acid" refers to single-stranded or double-stranded oligomers or polymers of deoxyribonucleotides, or ribonucleotide bases, or both. A nucleotide monomer consists of a nucleic acid base, a pentose sugar (e.g., ribose or 2'-deoxyribose, but not limited to these), and one to three phosphate groups. Typically, nucleic acids are formed by phosphodiester bonds between individual nucleotide monomers. In relation to the present invention, the term nucleic acid includes, but is not limited to, ribonucleic acid (RNA) molecules and deoxyribonucleic acid (DNA) molecules, as well as synthetic forms of nucleic acids involving other bonds [e.g., peptide nucleic acids as described in Nielsen et al. (Science 254:1497-1500, 1991)]. Typically, nucleic acids are single-stranded or double-stranded molecules composed of naturally occurring nucleotides. A single-stranded description of a nucleic acid also (at least partially) defines the sequence of the complementary strand. Nucleic acids may be single-stranded, double-stranded, or contain portions of both double-stranded and single-stranded sequences. The double-stranded nucleic acid molecules illustrated may have 3' or 5' overhangs and therefore do not need to be completely double-stranded over their entire length, but are likely to be so. The term nucleic acid includes chromosomes or chromosomal segments, vectors (e.g., expression vectors), expression cassettes, naked DNA or RNA polymers, primers, probes, cDNA, genomic DNA, recombinant DNA, cRNA, mRNA, tRNA, microRNA (miRNA), or small interfering RNA (siRNA). Nucleic acids can be, for example, single-stranded, double-stranded, or triple-stranded, and are not limited to any particular length. Unless otherwise indicated, a particular nucleic acid sequence includes or encodes a complementary sequence in addition to any explicitly indicated sequence.

[0302] Nucleic acids can be present throughout a cell, in a cell lysate, or in a partially purified or substantially pure form. A nucleic acid is considered “isolated” or “substantially pure” if it has been purified by standard techniques from other cellular components or other contaminants (e.g., other cellular nucleic acids or proteins).

[0303] The nucleic acid molecules of this disclosure can be obtained using standard molecular biological techniques, including but not limited to amplification and reverse transcription of RNA. For example, once a DNA fragment encoding a variable strand is obtained, this DNA fragment can be further manipulated using standard recombinant DNA techniques to convert, for example, a variable region gene into a full-length gene. In this manipulation, the DNA fragment encoding the variant is operably ligated to another DNA molecule or a fragment encoding another protein (e.g., a constant region or a flexible linker). The term "operably ligated," as used in this context, is intended to mean that the two DNA fragments are functionally ligated, for example, so that the amino acid sequence encoded by the two DNA fragments remains in frame, or so that the protein is expressed under the control of a desired promoter. By operably ligating the variable-coding DNA to another DNA molecule encoding a constant region, isolated DNA encoding a variable region (e.g., a variable alpha region and / or a variable beta region) can be converted into a full-length gene. For example, the sequences of human constant region genes related to TCRs or antibodies are known in the art, and DNA fragments containing this region can be obtained by standard PCR amplification.

[0304] Typically, the nucleic acid is a DNA molecule or an RNA molecule, which may be included in a suitable vector.

[0305] The first polypeptide and the second polypeptide described herein may be encoded by one nucleic acid molecule or two separate nucleic acid molecules.

[0306] Accordingly, expression vectors and host cells for producing the antigen-binding proteins or their functional fragments described herein are also provided herein.

[0307] In a third aspect, the present invention relates to a vector comprising nucleic acid according to a second aspect of the present invention.

[0308] The terms “vector,” “cloning vector,” and “expression vector” refer to a medium through which a DNA or RNA sequence (e.g., an exogenous gene) can be introduced into a host cell, thereby transforming the host and promoting the expression (e.g., transcription and translation) of the introduced sequence.

[0309] Various expression vectors can be used to express polynucleotides encoding antigen-binding proteins or their functional fragments. Both virus-based and non-virus-based expression vectors can be used to produce the antigen-binding proteins or their functional fragments described herein in mammalian host cells. Examples of non-viral vectors and systems include multiple plasmids, plasmids, cosmids, episomes, artificial chromosomes, phages, or viral vectors.

[0310] Such vectors may contain regulatory elements (e.g., promoters, enhancers, terminators, and the like) to induce or trigger the expression of the polypeptide upon administration to a target. Examples of promoters and enhancers used in expression vectors for animal cells include the initial promoter and enhancer for SV40 (Mizukami T. et al. 1987), the LTR promoter and enhancer for Moloney mouse leukemia virus (Kuwana Y et al. 1987), the promoter (Mason JO et al. 1985) and enhancer (Gillies SD et al. 1983) for antibody heavy chains, and the like.

[0311] For example, all suitable vectors known in the art for protein expression are nonviral vectors useful for the expression of polynucleotides and polypeptides described herein in mammalian (e.g., human or non-human) cells. Other examples of plasmids include replication plasmids containing an origin of replication, or integration plasmids (e.g., pUC, pcDNA, pBR, and the like).

[0312] The term "viral vector" refers to a nucleic acid vector construct that contains at least one element of viral origin, has the ability to be packaged into a viral vector particle, and encodes at least an exogenous nucleic acid. Vectors and / or particles can be used to introduce a desired nucleic acid into cells, either in vitro or in vivo. Numerous forms of viral vectors are known in the art. Useful viral vectors include retrovirus, lentivirus, adenovirus, adeno-associated virus, herpesvirus-based vectors, SV40, papillomavirus, Epstein-Barr virus, vaccinia virus vectors, and Semliki Forest virus (SFV)-based vectors. Recombinant viruses can be produced by techniques known in the art, for example, by transfecting packaging cells or by transient transfection with a helper plasmid or virus. Typical examples of viral packaging cells include PA317 cells, PsiCRIP cells, GPenv+ cells, and 293 cells. Detailed protocols for producing such replication-deficient recombinant viruses can be found, for example, in International Publication No. 95 / 14785, International Publication No. 96 / 22378, U.S. Patent No. 5,882,877, U.S. Patent No. 6,013,516, U.S. Patent No. 4,861,719, U.S. Patent No. 5,278,056, and International Publication No. 94 / 19478.

[0313] The nucleic acids encoding the first polypeptide and the second polypeptide described herein may be present in the same vector or in separate vectors.

[0314] In a fourth aspect, the present invention relates to a host cell comprising an antigen-binding protein according to the first aspect of the present invention, a nucleic acid according to the second aspect, or a vector according to the third aspect. This host cell may be transfected, infected, or transformed with the nucleic acid and / or vector according to the present invention.

[0315] The term "transformation" refers to the introduction of an "exogenous" gene, DNA, or RNA sequence into a host cell such that the host cell expresses the introduced gene or sequence and produces a desired substance (typically an antigen-binding protein or a functional fragment thereof, as described herein). A host cell that receives and expresses the introduced DNA or RNA is "transformed."

[0316] The nucleic acids of the present invention can be used to produce the recombinant antigen-binding protein of the present invention in a suitable expression system. The term "expression system" means, for example, a host cell and a suitable vector under suitable conditions for the expression of a protein encoded by foreign DNA that has been introduced into the host cell via a vector.

[0317] Common expression systems include Escherichia coli (E. coli) host cells and plasmid vectors, insect host cells and baculovirus vectors, and mammalian host cells and vectors. Other examples of host cells include, but are not limited to, prokaryotic cells (e.g., bacteria) and eukaryotic cells (e.g., yeast cells, mammalian cells, insect cells, plant cells, etc.). Specific examples include Escherichia coli (E. coli), yeast of the genera Kluyveromyces or Saccharomyces, mammalian cell lines (e.g., Vero cells, CHO cells, 3T3 cells, COS cells, HEK cells, etc.), and primary or established mammalian cell cultures (e.g., those produced from lymphoblasts, fibroblasts, germ cells, epithelial cells, nerve cells, adipocytes, etc.). Other examples include mouse SP2 / 0-Ag14 cells (ATCC CRL1581), mouse P3X63-Ag8.653 cells (ATCC CRL1580), CHO cells lacking the dihydrofolate reductase gene (Urlaub G et al; 1980), rat YB2 / 3HL.P2.G11.16Ag.20 cells (ATCC CRL1662), and similar cells. In some embodiments, YB2 / 0 cells may be preferred because, when expressed in these cells, the ADCC activity of the chimeric or humanized antibody is enhanced.

[0318] In one embodiment, the present invention refers to the antigen-binding protein of the present invention as defined herein above, or a nucleic acid encoding the antigen-binding protein of the present invention, or a vector encoding the antigen-binding protein of the present invention, wherein the host cell is preferably a) a lymphocyte, for example, a T lymphocyte or T lymphocyte precursor cell, for example, a CD4 or CD8 positive T cell, or b) a cell for recombinant expression, for example, a Chinese hamster ovary (CHO) cell.

[0319] In particular, for the expression of a portion of the antigen-binding protein of the present invention (especially an antigen-binding protein comprising two unlinked polypeptides), the expression vector may be of the type in which the gene encoding the antibody heavy chain and the gene encoding the antibody light chain are located in separate vectors, or of the type in which both genes are located in the same vector (tandem type). Tandem humanized antibody expression vectors are preferred in terms of ease of construction of the antigen-binding protein expression vector, ease of introduction into animal cells, and balance between the expression levels of the antibody H chain and L chain in animal cells (Shitara K et al. J Immunol Methods. 1994 Jan. 3; 167(1-2):271-8). Examples of tandem humanized antibody expression vectors include pKANTEX93 (International Publication No. 97 / 10354), pEE18, and similar types.

[0320] In one embodiment, such recombinant host cells can be used to produce at least one antigen-binding protein of the present invention.

[0321] Pharmaceutical composition In a fifth aspect, the present invention relates to a pharmaceutical composition comprising an antigen-binding protein of the present invention, a nucleic acid of the present invention, a vector of the present invention, or a host cell of the present invention, and a pharmaceutically acceptable carrier.

[0322] The antigen-binding protein of the present invention has been shown to induce cytotoxicity against tumor cells. Therefore, the antigen-binding protein of the present invention is useful for destroying tumor cells in patients. An immune response in patients can ideally be induced by direct administration of the described antigen-binding protein to the patient, combined with an immunogenicity-enhancing agent (i.e., an adjuvant). The immune response resulting from such therapeutic vaccination can be expected to be highly specific to tumor cells because the peptide SLLQHLIGL (SEQ ID NO: 50) is not presented at the same copy number or in excess on normal tissues, thus preventing the risk of an undesirable autoimmune response against the patient's normal tissue cells.

[0323] The present invention also relates to antigen-binding proteins according to the present invention for use as pharmaceuticals. The present invention also relates to pharmaceutical compositions according to the present invention for use as pharmaceuticals.

[0324] When used herein, the terms “pharmaceutical composition” or “therapeutic composition” refer to a compound or composition capable of inducing a desired therapeutic effect when appropriately administered to a subject.

[0325] In some embodiments, the subject may also be referred to as the patient.

[0326] Such therapeutic or pharmaceutical compositions may be mixtures with pharmaceutically or physiologically acceptable formulations selected to suit a mode of administration, and may further contain a therapeutically effective amount of the antigen-binding protein of the present invention, or the therapeutic agent.

[0327] The antigen-binding protein of the present invention is typically supplied as part of a sterile pharmaceutical composition, usually comprising a pharmaceutically acceptable carrier and / or a pharmaceutically acceptable carrier diluent.

[0328] "Pharmacologically" or "pharmaceutically acceptable" means, where applicable, a molecular entity or composition that, when administered to a mammal (in particular, a human), does not produce an adverse reaction, an allergic reaction, or any other adverse reaction. A pharmacopoeia is a pharmacopoeia of any type of non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation aid.

[0329] "Pharmacologically acceptable carriers or excipients" may also be referred to as "pharmaceutically acceptable diluents" or "pharmaceutically acceptable media," and may include physiologically compatible solvents, bulking agents, stabilizers, dispersion media, coating agents, antimicrobial and antifungal agents, isotonic and absorption retarders, and the like. In one embodiment, the carrier is an aqueous carrier.

[0330] In another embodiment, the aqueous carrier, when combined with the antigen-binding protein described herein, can confer improved properties (e.g., improved solubility, efficacy, and / or improved immunotherapy).

[0331] The form, route of administration, dosage, and regimen of the pharmaceutical composition naturally depend on the condition being treated, the severity of the disease, the patient's age, weight, and sex, etc. The pharmaceutical composition may be in any preferred form (depending on the desired method of administration to the patient). The pharmaceutical composition may be supplied in unit dosage forms, generally in sealed containers, and as part of a kit. Such a kit would typically (but not necessarily) include instructions for use. Such a kit may also contain multiple of the aforementioned unit dosage forms.

[0332] Empirical considerations, such as biological half-life, generally contribute to dosage determination. The frequency of administration may be determined and adjusted during the treatment period, based on a reduction in the number of cancer cells, maintenance of a reduction in cancer cells, reduction of cancer cell proliferation, or elimination of cancer cells. Alternatively, a sustained-release formulation of antigen-binding protein may be appropriate. Various formulations and devices for achieving sustained release are known in the art.

[0333] In one embodiment, the dose of the antigen-binding molecule may be determined empirically in an individual receiving one or more doses. The individual is administered an increasing dose of the antigen-binding protein. To evaluate the effectiveness of the antigen-binding protein, markers of cancer cell status may be tracked. This includes direct measurement of cancer cell proliferation and cell death by FACS, other imaging techniques, improvement in health status as assessed by such measurements, or improvement in quality of life or extension of survival as measured by accepted tests. It will be apparent to those skilled in the art that the dose will vary depending on the individual, the stage of the disease, and past and contemporary treatments being used.

[0334] In particular, the pharmaceutical composition contains a pharmaceutically acceptable medium for an injectable formulation. This may be, in particular, isotonic sterile saline (monosodium or disodium phosphate, sodium chloride, potassium chloride, calcium chloride, or magnesium chloride, and the like, or mixtures of such salts), or a dry (especially lyophilized) composition, which may, in some cases, constitute an injectable solution by the addition of sterile water or saline.

[0335] The dose used for administration can be adapted as a function of various parameters, particularly as a function of the mode of administration used, as a function of the associated pathology, or as a function of the desired duration of treatment.

[0336] For the preparation of a pharmaceutical composition, an effective amount of the antigen-binding protein of the present invention may be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.

[0337] Suitable pharmaceutical forms for injection include sterile aqueous solutions or dispersions; formulations containing sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. In all cases, the formulation must be sterile and fluid enough to be easily injected with a syringe. The formulation must be stable under manufacturing and storage conditions and protected from contamination by microorganisms such as bacteria and fungi.

[0338] A solution of the active compound, either as a free base or a pharmacologically acceptable salt, can be prepared in water appropriately mixed with a surfactant such as hydroxypropyl cellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycol, and mixtures thereof, and even in oil. Under normal storage and use conditions, these formulations contain preservatives to prevent microbial growth.

[0339] The antigen-binding protein of the present invention can be incorporated into a composition in neutral or salt form. Examples of pharmaceutically acceptable salts include acid addition salts (formed with the free amino group of the protein), which are formed with inorganic acids (e.g., hydrochloric acid or phosphoric acid) or organic acids (e.g., acetic acid, oxalic acid, tartaric acid, mandelic acid, and the like). Salts formed with free carboxyl groups can also be derived from inorganic bases (e.g., sodium, potassium, ammonium, calcium, or ferric hydroxide) and organic bases (e.g., isopropylamine, trimethylamine, glycine, histidine, procaine, and the like).

[0340] Sterile injectable solutions are prepared by incorporating the required amount of active compound, along with various other components listed above as needed, into a suitable solvent, followed by sterilization by filtration. Generally, dispersions are prepared by incorporating various sterilizing active ingredients into a sterilizing medium containing a basic dispersion medium and other necessary components from those listed above. In the case of sterilizing powders for preparing sterilizing injectable solutions, preferred preparation methods are vacuum drying and freeze-drying techniques, which yield powders of the active ingredients and any additional desired components from a pre-sterilized filtered solution.

[0341] The preparation of more or higher concentrations of solutions for direct injection is also being considered, and the use of DMSO as a solvent is expected to result in very rapid penetration, allowing high concentrations of the active ingredient to be delivered to small tumor areas.

[0342] In formulations, the solution is administered in a manner compatible with the administered formulation and in a therapeutically effective amount. This formulation can be easily administered in various dosage forms, such as the types of injection solutions described above, but drug-releasing capsules and similar forms may also be used.

[0343] Method for producing antigen-binding proteins In a sixth aspect, the present invention relates to a method for producing an antigen-binding protein according to a first aspect of the present invention, (a) Prepare the host cells, (b) Prepare a gene construct containing the coding sequence that encodes this antigen-binding protein. (c) Introducing the gene construct into the host cell, (d) Expressing the gene construct in the host cells, and as appropriate (e) Selecting cells that express and / or secrete the antigen-binding protein. Regarding methods including

[0344] In one embodiment, the method further comprises isolating and purifying antigen-binding proteins from host cells, and, as appropriate, reconstituting antigen-binding proteins in T cells. Those skilled in the art can easily select host cells suitable for expressing antigen-binding proteins.

[0345] The antigen-binding proteins of the present invention can be produced by any technique known in the art (for example, any chemical, biological, genetic, or enzymatic technique, either alone or in combination).

[0346] The antigen-binding protein of the present invention can be suitably separated from the culture medium by an antibody purification procedure (e.g., protein A-Sepharose, hydroxyl apatite chromatography, gel electrophoresis, dialysis, or affinity chromatography).

[0347] In one embodiment, recovering the expressed antigen-binding protein or polypeptide means, as herein it refers to performing protein A chromatography, kappa select chromatography, and / or size exclusion chromatography, preferably protein A chromatography and / or size exclusion chromatography, more preferably protein A chromatography and size exclusion chromatography.

[0348] By knowing the amino acid sequence of a desired sequence, those skilled in the art can produce the antigen-binding protein of the present invention by standard techniques for polypeptide production. For example, this protein can be synthesized using known solid-phase methods, particularly using commercially available peptide synthesizers (e.g., those from Applied Biosystems, Foster City, California), and according to the manufacturer's instructions. Alternatively, the antibody and antigen-binding protein of the present invention can be produced by recombinant DNA and hereditary transfection techniques known in the art [see Morrison SL. et al. (1984) and patent documents US5,202,238; and US5,204,244]. For example, a fragment can be obtained as a DNA expression product after incorporating the DNA sequence encoding the desired (poly)peptide into an expression vector and introducing such a vector into a suitable eukaryote or prokaryote host expressing the desired polypeptide, and then this fragment can be isolated using known techniques.

[0349] In one example [i.e., in the case of TCER®], DNA sequences encoding various combinations of VH and VL, as well as variable alpha (V-alpha) and variable beta (V-beta), and DNA sequences encoding the linker can be obtained, for example, by gene synthesis. The obtained DNA sequences can be cloned in-frame into expression vectors encoding the hinge region, CH2 domain, and CH3 domain, respectively, derived from human IgG4 [Accession#:K01316] and IgG1 [Accession#:P01857], and can be further manipulated. By performing the operation according to the method described by Reiter et al. (Stabilization of the Fv Fragments in Recombinant Immunotoxins by Disulfide Bonds Engineered into Conserved Framework Regions. Biochemistry, 1994, 33, 5451 - 5459), a knob-into-hole mutation can be incorporated into the CH3 domain with or without additional interchain disulfide bond stabilization; or the N-glycosylation site in CH2 can be removed (e.g., N297Q mutation); respectively, V L and V H F c Silencing mutations or additional disulfide bond stabilizations may be introduced.

[0350] Methods for producing humanized antibodies based on conventional recombinant DNA and gene transfection techniques are known in the art (see, for example, Riechmann L. et al. 1988; Neuberger MS. et al. 1985) and can be readily applied to the production of antigen-binding proteins.

[0351] In one example, the vector for the expression of the recombinant antigen-binding protein of the present invention was designed as a monocistronic vector controlled by, for example, an HCMV-derived promoter element and a pUC19 derivative. Plasmid DNA was grown in E. coli (E. coli) according to a standard culture method, and then purified using a commercially available kit (Macherey & Nagel). The purified plasmid DNA was used for transient transfection of CHO-S cells, for example, according to the manufacturer's instructions (ExpiCHO® system; Thermo Fisher Scientific). The transfected CHO cells were cultured, for example, at 32°C to 37°C for 6 to 14 days and fed with ExpiCHO® Feed solution 1 to 2 times.

[0352] The conditioned cell supernatant was clarified by filtration (0.22 μm) using, for example, a Sartoclear Dynamics® Lab Filter Aid (Sartorius). Bispecific antigen-binding proteins were purified using, for example, an Aekta Pure 25 L FPLC system (GE Lifesciences) equipped for in-line affinity chromatography and size exclusion chromatography. Affinity chromatography was performed, for example, using a Protein A or L column (GE Lifesciences) according to a standard affinity chromatography protocol. Size exclusion chromatography was performed, for example, using a Superdex 200 pg 16 / 600 column (GE Lifesciences) according to a standard protocol, immediately after elution from the affinity column (pH 2.8) to obtain high-purity monomeric proteins. Protein concentrations were determined using a NanoDrop system (Thermo Scientific) using, for example, a decay coefficient calculated according to the predicted protein sequence. Concentrations were adjusted as needed using a Vivaspin device (Sartorius). Finally, the purified molecules were stored in phosphate-buffered saline at a concentration of approximately 1 mg / mL at a temperature of 2–8°C, for example.

[0353] The quality of the purified bispecific antigen-binding proteins was determined, for example, by HPLC-SEC using a MabPac SEC-1 column (5 μm, 7.8 × 300 mm) operating in a Vanquish UHPLC-System with 50 mM sodium phosphate (pH 6.8) containing 300 mM NaCl.

[0354] Treatment methods and therapeutic uses In a seventh aspect, the present invention provides an antigen-binding protein of a first aspect, a nucleic acid of a second aspect, a vector of a third aspect, a host cell of a fourth aspect, or a pharmaceutical composition of a fifth aspect for use in pharmaceuticals, particularly for use in the diagnosis, prevention, and / or treatment of proliferative disorders. For therapeutic use (i.e., prevention and / or treatment), the antigen-binding protein has a first antigen-binding site (i.e., V) that binds to the PRAME antigen peptide in a complex with MHC. A and V B (Antigen binding site formed by) and a second antigen binding site that binds to the antigen of effector cells (i.e., V L and V H Preferably, the compound includes an antigen-binding site formed by [the compound]. The inventors have demonstrated the cytotoxic activity of constructs of several bispecific compounds of the present invention against PRAME-positive cancer cell lines such as Hs695T and U20S in the in vitro experimental section. The inventors have further demonstrated that the cytotoxic activity is highly specific and limited to PRAME-positive cells, as only slight lysis induced by the bispecific antigen-binding protein was observed in cell lines that did not display the peptide PRAME-004.

[0355] Therefore, cancer can be treated using the antigen-binding proteins of the present invention [particularly bispecific antigen-binding proteins such as TCER®]. The antigen-binding proteins of the present invention can be used for therapeutic purposes in humans and / or non-human mammals. In one embodiment, the antigen-binding protein of the present invention can bind to tumor cells and reduce the proliferation of tumor cells that present the peptide SLLQHLIGL (SEQ ID NO: 50):MHC complex on their cell surface, and / or kill them. It is understood that this antigen-binding protein is administered at concentrations that promote binding under physiological (e.g., in vivo) conditions. In another embodiment, the antigen-binding protein can be used in immunotherapy against tumor cells in various tissues such as the lungs, breasts, ovaries, or kidneys. In yet another embodiment, this antigen-binding protein can bind to tumor cells on its own and reduce the proliferation of these tumor cells, and / or kill them.

[0356] Accordingly, the present invention relates to a method for treating or preventing proliferative disorders, comprising administering a therapeutically effective amount of an antigen-binding protein, nucleic acid, or vector, host cell, or pharmaceutical composition according to the present invention, as defined in the "antigen-binding protein," "nucleic acid," or "pharmaceutical composition" sections of this specification, to a target subject.

[0357] In certain embodiments, the present invention relates to a method for treating a subject having a proliferative disorder, comprising administering to the subject T cells expressing the antigen-binding protein of the present invention on the cell surface.

[0358] In further embodiments, the present invention refers to a method for inducing an immune response in a subject having a proliferative disorder, comprising administering to the subject a composition comprising T cells expressing an antigen that recognizes the construct of the present invention on its cell surface.

[0359] In one embodiment, the immune response referred to in the method is a cytotoxic T cell response.

[0360] In one embodiment, the antigen-binding protein of the present invention, the nucleic acid of the present invention, or the vector of the present invention, the host cell of the present invention, or the pharmaceutical composition of the present invention are intended for use in the diagnosis, prevention, and / or treatment of proliferative disorders.

[0361] The present invention further refers to the use of antigen-binding proteins, nucleic acids or vectors, host cells or pharmaceutical compositions according to the present invention for the preparation of pharmaceuticals for treating or preventing target proliferative disorders or disorders.

[0362] In one embodiment, the present invention refers to a method for inducing an immune response in a patient having cancer that presents a peptide comprising or consisting of the amino acid sequence of SLLQHLIGL (SEQ ID NO: 50) in complex with an MHC protein, comprising administering the patient the antigen-binding protein of the present disclosure, wherein the cancer is selected from the group of cancers consisting of acute myeloid leukemia, breast cancer, cholangiocarcinoma, gallbladder cancer, glioblastoma, hepatocellular carcinoma, head and neck squamous cell carcinoma, melanoma, achromatic melanoma, non-Hodgkin lymphoma, non-small cell lung cancer adenocarcinoma, non-small cell lung cancer, squamous cell non-small cell lung cancer, ovarian cancer, esophageal cancer, renal cell carcinoma, small cell lung cancer, bladder cancer, uterine and endometrial cancer, osteosarcoma, chronic lymphocytic leukemia, colorectal cancer, and synovial sarcoma.

[0363] In one embodiment, the present invention refers to the use of an antigen-binding protein, nucleic acid or vector, host cell or pharmaceutical composition according to the present invention for treating or preventing a target disease or disorder.

[0364] The terms “subject” or “individual” are used interchangeably and can be, for example, human or non-human mammal, preferably human.

[0365] In relation to the present invention, the terms “to treat” or “treatment” refer to therapeutic use (i.e., on an object having a given disease) and mean reversal, alleviation, or inhibition of the progression of one or more symptoms of such disorder or condition. Thus, treatment refers not only to treatments that result in a complete cure of the disease, but also to treatments that delay the progression of the disease and / or extend the lifespan of the object.

[0366] "To prevent" means preventative use (i.e., for a subject that is susceptible to developing a given disease).

[0367] In one embodiment, “disease” or “disorder” means any condition that would benefit from treatment with the antigen-binding protein of the present invention. In one embodiment, this includes chronic and acute disorders or diseases, including pathological conditions that make a subject susceptible to the disorder in question. The term “requiring treatment” refers to subjects that already have a disorder, and subjects for whom prevention of the disorder should be sought.

[0368] In certain embodiments, the antigen-binding protein of the present invention is bispecific and, more specifically, is TCER® as described herein.

[0369] "Proliferative disorders" such as cancer are characterized by the uncontrolled and / or inappropriate proliferation of cells.

[0370] Therefore, in one embodiment, the proliferative disorder is cancer.

[0371] In further embodiments, the cancer is characterized by the overexpression, mutation, and / or presentation of MHC-associated tumor-related antigens derived from PRAME.

[0372] Therefore, the most preferred type of cancer is PRAME-positive cancer.

[0373] In connection with the present invention, a cancer is considered "PRAME-positive" if the relevant peptide (e.g., PRAME-004 peptide) is presented in more than 98% of all cancers according to NCI guidelines. In all other indications listed herein, biopsy may be performed as is standard practice in the treatment of these cancers, and the peptide may be identified according to XPresident® and related methods [International Publication No. 03 / 100432; International Publication No. 2005 / 076009; International Publication No. 2011 / 128448; International Publication No. 2016 / 107740, U.S. Patent No. 7,811,828, U.S. Patent No. 9,791,444, and U.S. Patent Application Publication No. 2016 / 0187351 (each of which is incorporated herein by reference in its entirety)]. In one embodiment, for example, cancer can be easily assayed (i.e., diagnosed) by using the antigen-binding protein of the present invention. Methods for identifying cancers that express antigens using antigen-binding proteins are known to those skilled in the art. It should be understood that the terms “cancer” and “carcinoma” are not interchangeable herein, as carcinoma is a specific type of cancer that appears in the skin or tissues that reinforce or cover organs of the body.

[0374] In one embodiment, the cancers that are PRAME "positive" (i.e., present the target peptide) are selected from the group consisting of acute myeloid leukemia, breast cancer, cholangiocarcinoma, gallbladder cancer, glioblastoma, hepatocellular carcinoma, head and neck squamous cell carcinoma, melanoma, achromatic melanoma, non-Hodgkin lymphoma, non-small cell lung cancer adenocarcinoma, non-small cell lung cancer, squamous cell non-small cell lung cancer, ovarian cancer, esophageal cancer, renal cell carcinoma, small cell lung cancer, bladder cancer, uterine and endometrial cancer, osteosarcoma, chronic lymphocytic leukemia, colorectal cancer, and synovial sarcoma, preferably selected from the group consisting of breast cancer, cholangiocarcinoma, hepatocellular carcinoma, head and neck squamous cell carcinoma, squamous cell non-small cell lung cancer, ovarian cancer, esophageal cancer, renal cell carcinoma, small cell lung cancer, bladder cancer, uterine and endometrial cancer, and synovial sarcoma.

[0375] In one embodiment, if the cancer is one in which the PRAME antigen is overexpressed, mutated, and / or presents a tumor-associated antigen derived from PRAEM related to MHC, it can be easily assayed, for example, by using the antigen-binding protein of the present invention. Methods for identifying cancers expressing antigens using antigen-binding proteins are known to those skilled in the art.

[0376] A text outlining guidelines for cancer treatment is *Cancer, Principles and Practice of Oncology, 4th Edition*, DeVita et al, Eds. JB Lippincott Co., Philadelphia, Pa. (1993). As recognized in the relevant field, the appropriate treatment approach is selected according to the specific type of cancer and other factors (e.g., the patient's overall condition). The antigen-binding protein of the present invention can be used on its own or added to treatment regimens using other antineoplastic agents in the treatment of cancer patients.

[0377] Accordingly, in some embodiments, for example, antigen-binding proteins may be administered simultaneously with, before, or after, various drugs and treatments widely used in cancer treatment, such as chemotherapeutic agents, non-chemotherapeutic agents, antineoplastic agents, and / or radiation.

[0378] In one embodiment, the present invention relates to a method for treating a patient having cancer that presents a peptide containing or comprising the amino acid sequence of SLLQHLIGL (SEQ ID NO: 50) in complex with an MHC protein, comprising administering the patient an antigen-binding protein of the present disclosure, wherein the cancer is selected from the group of cancers consisting of acute myeloid leukemia, breast cancer, cholangiocarcinoma, gallbladder cancer, glioblastoma, hepatocellular carcinoma, head and neck squamous cell carcinoma, melanoma, achromatic melanoma, non-Hodgkin lymphoma, non-small cell lung cancer adenocarcinoma, non-small cell lung cancer, squamous non-small cell lung cancer, ovarian cancer, esophageal cancer, renal cell carcinoma, small cell lung cancer, bladder cancer, uterine and endometrial cancer, osteosarcoma, chronic lymphocytic leukemia, colorectal cancer, and synovial sarcoma.

[0379] In this specification, "diagnosis" refers to a medical diagnosis, which means determining which disease or condition explains a person's symptoms and signs.

[0380] The “therapeutably effective amount” of an antigen-binding protein or its pharmaceutical composition means an amount of antigen-binding protein sufficient to treat the proliferative disorder with a reasonable benefit / risk ratio applicable to any medical treatment. However, it will be understood that the total daily dose of the antigen-binding protein, nucleic acid or vector, host cell or pharmaceutical composition of the present invention is to be determined by the attending physician within the bounds of sound medical judgment. The specific therapeutically effective dose for any particular patient depends on a variety of factors, including: the disorder being treated and its severity; the activity of the specific antigen-binding protein being employed; the specific composition being employed; the patient’s age, weight, overall health, sex, and diet; the timing, route of administration, and elimination rate of the specific polypeptide being employed; the duration of treatment; any drugs used in combination with or concurrently with the specific polypeptide being employed; and similar factors known in the medical art. For example, it is known to those skilled in the art to start with a dose of the compound at a level lower than the level required to obtain the desired therapeutic effect and to gradually increase the dose until the desired effect is achieved.

[0381] In one embodiment, the effectiveness of treatment with the antigen-binding protein of the present invention is assayed in vivo, for example, in a mouse model of cancer, by measuring the change in tumor volume between the treatment group and the control group.

[0382] The pharmaceutical compositions, vectors, nucleic acids, and cells of the present invention may be provided in substantially pure form, for example, in a form that is 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% pure.

[0383] The antigen-binding protein of the present invention, the nucleic acid of the present invention, or the vector of the present invention, the host cell of the present invention, or the pharmaceutical composition of the present invention may be administered by any feasible method.

[0384] As disclosed herein, in some embodiments, the host cells defined above herein are used in the medical applications or treatment methods described herein. In the same embodiments, the host cells are preferably a) lymphocytes, for example, T lymphocytes or T lymphocyte precursor cells, for example, CD4 or CD8 positive T cells, and most preferably T cells.

[0385] Therefore, the host cells (preferably T cells) of the present invention can be used as an active ingredient in a therapeutic composition. Accordingly, the present invention also provides a method for killing target cells in a patient in which the target cells abnormally express a polypeptide comprising the peptide SLLQHLIGL (SEQ ID NO: 50), the method comprising administering an effective number of host cells (preferably T cells) to the patient. In connection with this method, the host cells, upon administration to the subject, preferably induce an immune response.

[0386] In some embodiments, a TCR-induced response, or T cell response, may refer to the enhancement and activation of effector function induced by peptides such as SLLQHLIGL (SEQ ID NO: 50), either in vitro or in vivo. In the case of MHC class I-restricted cytotoxic T cells, for example, effector function may include peptide-pulsed, peptide precursor-pulsed, or naturally peptide-presenting target cell lysis, peptide-induced cytokine secretion (preferably interferon-gamma, TNF-alpha, or IL-2), peptide-induced effector molecule secretion (e.g., granzyme or perforin), or degranulation.

[0387] Therefore, the host cells defined above in this specification may be derived from the subject (self) or from another individual, preferably the other individual being healthy.

[0388] "Healthy" means that the subject is in generally good health, preferably has a qualified immune system, and more preferably is free from any disease that can be readily tested and detected.

[0389] In a specific example, the host cell is a T cell. Therefore, in relation to the present invention, when T cells as defined above herein are used as pharmaceuticals, the T cells are usually harvested from cells by apheresis. The T cells are then genetically engineered to express the antigen-binding protein of the present invention on their cell surface, the genetically engineered T cells are then grown, and then reinjected into the subject. In this example, the antigen-binding protein is preferably a TCR.

[0390] In an alternative approach, the host cell may be a stem cell, such as a mesenchymal stem cell, that has been engineered to express the antigen-binding protein of the present invention. In this example, the antigen-binding protein is a soluble protein such as an antibody, scTCR, or diabody as defined above herein.

[0391] Accordingly, host cells are transfected, infected, or transformed with nucleic acids and / or vectors according to the present invention, as described in the “Nucleic Acids, Vectors, and Recombinant Host Cells” section above in this specification.

[0392] When host cells are transfected to express the antigen-binding protein of the present invention, the cells preferably contain an expression vector capable of expressing the antigen-binding protein. The host cells can then be referred to as activated host cells.

[0393] This so-called T cell adoptive transfer protocol is publicly known in the field. An overview can be found in Gattinoni et al. and Morgan et al. [Gattinoni, L. et al., Nat. Rev. Immunol. 6 (2006): 383-393; Morgan, RA et al., Science 314 (2006): 126-129].

[0394] T cells can be generated in vitro using many other methods. For example, autologous tumor-infiltrating lymphocytes can be used to generate CTLs. Plebanski et al. (Plebanski, M. et al., Eur.J Immunol 25 (1995): 1783-1787) used autologous peripheral blood lymphocytes (PLBs) in the preparation of T cells. Similarly, B cells can be used in the production of autologous T cells.

[0395] Allogeneic cells may also be used in the preparation of T cells, and the method is described in detail in U.S. Patent No. 6,805,861, which is incorporated herein by reference.

[0396] Host cells expressing the antigen-binding protein of the present invention against the peptide SLLQHLIGL (SEQ ID NO: 50) are useful for therapy. Therefore, a further aspect of the present invention provides activated host cells obtained by the method described above.

[0397] Activated host cells produced by the above method can specifically recognize cells that abnormally express polypeptides containing the peptide SLLQHLIGL (SEQ ID NO: 50).

[0398] "Abnormally expressed" also means that the polypeptide is overexpressed compared to the expression level in normal (healthy) tissue, or that the gene is silent in the tissue from which the tumor originates but is expressed within the tumor. "Overexpressed" means that the polypeptide is present at a level at least 1.2 times the level present in normal tissue, preferably at a level at least 2 times, more preferably at least 5 or 10 times, the level present in normal tissue.

[0399] In one embodiment, host cells (particularly T cells) recognize the cells by interacting with (e.g., binding to) the PRAME-004 complex via their antigen-binding proteins (particularly their TCRs). The host cells are useful in a method of killing target cells in a patient whose target cells abnormally express a polypeptide containing the peptide SLLQHLIGL (SEQ ID NO: 50), and the patient is administered an effective number of activated host cells. The T cells administered to the patient may be derived from the patient and may be activated as described above (i.e., autologous T cells). Alternatively, the T cells may not be derived from the patient but from another individual. Naturally, this is preferable when the individual is a healthy individual. By “healthy individual,” we mean that the individual is in generally good health, preferably has a qualified immune system, and more preferably is free from any disease that can be readily tested and detected.

[0400] In vivo, the target cells of CD8-positive T cells according to the present invention may be tumor cells (which may express MHC class II) and / or stromal cells (which may also express MHC class II) surrounding the tumor (tumor cells) [Dengjel, J. et al., Clin Cancer Res 12 (2006): 4163-4170].

[0401] Diagnostic use PRAME is expressed on the surface of PRAME-expressing cancer as defined above herein. The antigen PRAME constitutes a cancer marker and therefore may be used to indicate the effectiveness of anti-cancer treatment or to detect disease recurrence.

[0402] Therefore, in another embodiment, the present invention provides an antigen-binding protein of a first embodiment, a nucleic acid of a second embodiment, a vector of a third embodiment, a host cell of a fourth embodiment, or a pharmaceutical composition of a fifth embodiment for use as a diagnostic agent, particularly for use as an in vivo diagnostic agent. In a preferred embodiment, the diagnostic agent is for the diagnosis of proliferative disorders. In a more preferred embodiment, the diagnostic agent is for the diagnosis of cancer, presenting a peptide containing or consisting of the amino acid sequence of SLLQHLIGL (SEQ ID NO: 50) in complex with an MHC protein, preferably the cancer being selected from the group of cancers consisting of acute myeloid leukemia, breast cancer, cholangiocarcinoma, gallbladder cancer, glioblastoma, hepatocellular carcinoma, head and neck squamous cell carcinoma, melanoma, achromatic melanoma, non-Hodgkin lymphoma, non-small cell lung cancer adenocarcinoma, non-small cell lung cancer, squamous cell non-small cell lung cancer, ovarian cancer, esophageal cancer, renal cell carcinoma, small cell lung cancer, bladder cancer, uterine and endometrial cancer, osteosarcoma, chronic lymphocytic leukemia, colorectal cancer, and synovial sarcoma.

[0403] Those skilled in the art will know that for diagnostic purposes, this antigen-binding protein is V A and V B It includes, but preferably V L and V H I am aware that it does not include

[0404] In some embodiments, the antigen-binding protein of the present invention is used as a component of an assay related to therapies targeting tumors expressing PRAME to determine a patient's sensitivity to a therapeutic agent, to monitor the effectiveness of an anti-cancer treatment, or to detect disease recurrence after treatment. In particular, V as defined herein A Domain and V BAntigen-binding proteins containing domains are used as components of diagnostic assays, while bispecific antigen-binding proteins are used as components of therapeutic drugs.

[0405] Therefore, a further object of the present invention relates to an antigen-binding protein according to the present invention for use in detecting PRAME expression in a subject in vivo, or for detecting PRAME expression in a biological sample of a subject ex vivo or in vitro. The detection may, in particular, include: a) Diagnosing the presence of cancer in the subject, b) Determining the sensitivity of cancer patients to PRAME-targeting therapies, or c) By detecting the expression of the surface protein PRAME on tumor cells, in particular with respect to the bispecific therapy according to the present invention, the effectiveness of anti-PRAME cancer therapy or the detection of cancer recurrence after anti-PRAME cancer therapy.

[0406] In some embodiments, this antigen-binding protein is intended for use in vitro or ex vivo.

[0407] kit Finally, the present invention also provides a kit comprising at least one antigen-binding protein of the present invention.

[0408] In one embodiment, the kit is a) At least one antigen-binding protein of the present invention as defined in the “antigen-binding protein” section above, b) Packaging materials as appropriate, and c) A label or packaging insert included in the packaging material, which appropriately indicates that the antigen-binding protein is effective for the treatment of cancer or is effective for use in the treatment of cancer. Includes.

[0409] In related embodiments, at least one antigen-binding protein of the present invention is contained in single-chamber and / or multi-chamber pre-filled syringes [e.g., liquid syringes and lyosyringes].

[0410] In one embodiment, the present invention comprises a kit for manufacturing single-dose dosage units.

[0411] Accordingly, in one embodiment, the at least one antigen-binding protein of the present invention referred to in a) of the kit of the present invention is the dried antigen-binding protein of the present invention contained in a first container. The kit then further comprises a second container containing an aqueous formulation.

[0412] Therefore, in one embodiment, the kit is a) A first container containing at least one of the dried antigen-binding proteins of the present invention as defined in the “Antigen-binding proteins” section of this specification, b) A second container containing the aqueous formulation, c) Packaging materials as appropriate, and d) A label or packaging insert contained in the packaging material that indicates, as appropriate, that the antigen-binding protein is effective for the treatment of cancer or is effective for use in the treatment of cancer. Includes.

[0413] Aqueous formulations are typically aqueous solutions containing a pharmaceutically acceptable carrier as defined in the “Pharmaceutical Compositions” section above.

[0414] In related embodiments, “first container” and “second container” refer to the chambers of a multi-chamber pre-filled syringe (e.g., a lyophilized syringe).

[0415] Throughout this application, the term "and / or" is a grammatical conjunction that should be interpreted as encompassing the possibility of one or more of the cases in which it connects occurring. For example, the phrase "such native sequence proteins may be prepared using standard recombinant and / or synthetic methods" indicates that native sequence proteins may be prepared using standard recombinant and synthetic methods, or that native sequence proteins may be prepared using standard recombinant methods, or that native sequence proteins may be prepared using synthetic methods.

[0416] Furthermore, throughout this application, the term “including” should be interpreted as encompassing not only all features specifically mentioned, but also any, additional, or unspecified features. Where used herein, the use of the term “including” also discloses embodiments in which features other than those specifically mentioned are absent (i.e., “consisting of”).

[0417] Furthermore, the indefinite article "a" or "an" does not exclude the plural. The mere fact that certain means are enumerated in different dependent claims does not indicate that combinations of these means cannot be used advantageously.

[0418] The present invention will now be described in more detail with reference to the following drawings and examples. All documents and patent documents cited herein are incorporated herein by reference. Although the present invention is described in detail below, the examples should be considered illustrative and not limiting. [Examples]

[0419] [Example 1]

[0420] Single-stranded TCR (scTCR format) [Example 1.1] stable scTCR generation In this invention, TCR R11P3D3 (sequences 1 and 2, full length) was converted to a single-stranded TCR construct (scTCR R11P3D3, sequence 5) using variable alpha (sequence number 3) and beta (sequence number 4) domains, as well as a suitable glycine-serine linker sequence (sequence number 61). For TCR maturation via yeast surface presentation, DNA of the corresponding sequence was synthesized and transformed into a pCT302-based yeast display vector (yeast display vector) of Saccharomyces cerevisiae EBY100 (MATa AGA1::GAL1-AGA1::URA3 ura3-52 trp1 leu2-delta200 his3-delta200 pep4::HIS3 prbd1.6R can1 GAL) (ATCC(registered trademark) MYA-4941(trademark)) (Boder and Wittrup, Methods Enzymol. 2000;328:430-44;). The fusion protein (SEQ ID NO: 325) obtained after homologous recombination in yeast contains the leader peptide at the N-terminus of the Aga2p protein (SEQ ID NO: 88) (Boder and Wittrup, Nat Biotechnol. 1997 Jun;15(6):553-7), the target protein (i.e., scTCR R11P3D3 (SEQ ID NO: 5) or its variant, and additional peptide tags [FLAG and Myc (SEQ ID NOs: 99 and 288)] to determine the expression level of the fusion protein). A library of scTCR variants was prepared by PCR using degenerate primers, and yeast cells were transformed as described in International Publication No. 2018 / 091396, with 10 per library. 9 We obtained several yeast clones.

[0421] The selection process for yeast clones containing mutant scTCR variants with improved binding to PRAME-004 in relation to HLA-A*02 was essentially carried out as described by Smith et al. (Methods Mol Biol. 2015;1319:95-141). Expression determined by Myc-tag-FITC staining, particularly functional binding by HLA-A*02 / PRAME-004 tetramer staining, was applied to select the most promising candidates (Figure 1). Yeast surface presentation of scTCR transformation revealed nine framework mutations combined with three single-point CDR mutations, resulting in the stabilized scTCR R11P3D3SD (SEQ ID NO: 6) exhibiting improved expression and HLA-A*02 / PRAME-004 tetramer binding. [Example 1.2]

[0422] Evaluation of affinity maturation, binding motif, and specificity of stabilized scTCRs To generate scTCR molecules with higher binding affinity to HLA-A*02 / PRAME-004, all CDRs were individually matured using the previously identified stabilized scTCR R11P3D3SD (SEQ ID NO: 6). CDR residues were randomized using degenerate DNA oligo primers, essentially as described previously (Smith et al., Methods Mol Biol. 2015;1319:95-141). The resulting DNA library was transformed as described in Example 1.

[0423] To select specific R11P3D3SD scTCR mutants with enhanced affinity, the concentration of HLA-A*02 / PRAME-004 tetramers or monomers was reduced in each selection round. After four selection rounds, single scTCR clones were isolated and sequenced to obtain many affinity-mature CDR sequences. A strong improvement in HLA-A*02 / PRAME-004 monomer binding was demonstrated for scTCRs with mature CDRa1 sequences (SEQ ID NOs. 16-28, Figure 2), and also for scTCRs with mature CDRa2 and CDRb2 sequences (SEQ ID NOs. 29-32 and 35-45, Table 3). The selectivity for HLA-A*02 / PRAME-004 binding was maintained during maturation, as evidenced by the low binding of scTCRs to mixes of HLA-A*02 tetramers containing peptides with a high degree of sequence similarity to the PRAME-004 peptide (similar peptides or SimPep). All selected scTCR mutants showed substantial staining with the HLA-A*02 / PRAME-004 monomer at a concentration of 10 nM, whereas the immature, stabilized scTCR R11P3D3SD, used as a reference, did not stain (Figure 2 and Table 3). Furthermore, binding of mature scTCRs to a mix of similar peptides applied in a highly binding-active format of the HLA-A*02 tetramer at a concentration of 10 nM was either undetectable or showed only a weak signal compared to HLA-A*02 / PRAME-004 monomer binding, supporting the ability of mature scTCR mutants to bind to PRAME-004 target peptides with high specificity.

[0424] [Table 3]

[0425] To further increase the affinity of scTCR clones, mature CDRs identified in the CDR library described above were systematically combined into a single DNA library and transformed into Saccharomyces cerevisiae EBY100 as described in Example 1.1. This library was selected using the HLA-A*02 / PRAME-004 monomer, and scTCRs from single yeast clones were sequenced and analyzed for binding to the HLA-A*02 monomer containing either the PRAME-004 target peptide or one peptide from a group of 26 peptides (similar peptides) (SEQ ID NOs. 51-60, 62-69, and 71-78) that share sequence similarity with PRAME-004. All selected high-affinity scTCR variants (SEQ ID NOs. 79-87, and 89-92) had low nanomolar or sub-nanomolar bound EC values ​​when calculated by nonlinear four-point curve fitting. 50 The values ​​indicated strong binding to the HLA-A*02 / PRAME-004 monomer (Table 4). With the exception of SMARCD1-001 (SEQ ID NO: 76), which induced a binding signal slightly above the background (Figure 4), none of the scTCR variants (SEQ ID NOs: 79-87 and 89-92) showed binding above the background level to any of the similar peptides (SEQ ID NOs: 51-60, 62-69, and 71-78) related to the HLA-A*02 monomer when applied at a concentration of 100 nM (Figures 3, 4, and Table 4). The presented data support the high binding specificity of scTCR variants combined with CDR mutations, and this binding characteristic was superior to that of the reference scTCR (R16P1C10_CDR6_scTCR, SEQ ID NO: 357), which showed strong binding to IFT17-003 (SEQ ID NO: 60) at a level indistinguishable from PRAME-004 binding (Figure 4).

[0426] A set of high-affinity scTCRs selected from yeast surface presentation were further investigated in relation to functional epitopes on target peptides associated with HLA-A*02 presentation, known as binding motifs. This was addressed by evaluating the binding of scTCR-containing yeast cells to single alanine substitutions at positions 1, 3, 4, 5, 6, 7, and 8 of the PRAME-004 target peptide (SEQ ID NOs. 318-324) and to each PRAME-004 peptide variant associated with HLA-A*02. High-affinity scTCR-containing yeast cells were stained with HLA-A*02 monomers and PRAME-004 or their respective alanine-substituted peptides at four concentrations (10 nM, 3 nM, 1 nM, 0.3 nM). Extensive binding motifs were revealed for all scTCR variants strongly recognized at positions 3, 5, and 7, as evidenced by the absence of staining signals at all monomer concentrations tested. Regarding positions 6 and 8 of the PRAME-004 peptide, a contribution to the binding motif can be assumed because alanine substitution at these positions significantly reduced the staining signal, even when observed with lower stringency compared to positions 3, 5, and 7. Regarding positions 1 and 4 of the PRAME-004 peptide, it can be determined that the contribution to the binding motif is minimal or nonexistent because alanine substitution resulted in staining intensity nearly equivalent to that observed with the PRAME-004 target peptide (Figure 5 and Table 4).

[0427] For further analysis, five scTCR clones—R11P3D3SDA7_A02_scTCR (SEQ ID NO: 79), R11P3D3SDA7_A09_scTCR (SEQ ID NO: 82), R11P3D3SDA7_A10_scTCR (SEQ ID NO: 83), R11P3D3SDA7_B03_scTCR (SEQ ID NO: 85), and R11P3D3SDA7_B06_scTCR (SEQ ID NO: 87)—were converted to the scTCR-Fab bispecificity format to determine further protein characteristics (see the examples below).

[0428] [Table 4] [Example 2]

[0429] Production and Characterization of Soluble scTCR-Fab Molecules A TCR consisting of the V-alpha domain and V-beta domain was designed and tested in a single-stranded (scTCR) format coupled with a Fab fragment of a humanized UCHT1 antibody (Tables 5 and 18). A vector for recombinant protein expression was designed as a monocistronic vector controlled by an HCMV-derived promoter element pUC19 derivative. Plasmid DNA was amplified in Escherichia coli (E. coli) according to standard culture procedures and then purified using a commercially available kit (Macherey & Nagel). The purified plasmid DNA was used for transient transfection of CHO cells. Transfected CHO cells were cultured at 32°C–37°C for 10–11 days.

[0430] The conditioned cell supernatant was clarified by filtration (0.22 μm) using a Sartoclear Dynamics® Lab Filter Aid (Sartorius). Bispecific molecules were purified using an Aekta Pure 25 L FPLC system (GE Lifesciences) equipped for in-line affinity chromatography and size exclusion chromatography. Affinity chromatography was performed on a Protein L column (GE Lifesciences) according to a standard affinity chromatography protocol. Size exclusion chromatography was performed immediately after elution from the affinity column (pH 2.8) using a Superdex 200 pg 16 / 600 column (GE Lifesciences) according to a standard protocol to obtain high-purity monomeric proteins. Protein concentrations were determined using a NanoDrop system (Thermo Scientific) with decay coefficients calculated according to the predicted protein sequence. Concentrations were adjusted using a Vivaspin device (Sartorius) as needed. Finally, the purified molecules were stored in phosphate-buffered saline at a concentration of approximately 1 mg / mL at a temperature of 2–8°C. The yield of the final product was calculated after the completion of purification and formulation.

[0431] The quality of the purified bispecific molecules was determined by HPLC-SEC using a MabPac SEC-1 column (5 μm, 4 × 300 mm) operating in a Vanquish uHPLC-System with 50 mM sodium phosphate (pH 6.8) containing 300 mM NaCl.

[0432] Stress stability testing was performed by incubation of the formulation molecules in PBS at 40°C for up to two weeks. Integrity, aggregate content, and monomer recovery were analyzed by HPLC-SEC analysis as described above.

[0433] [Table 5]

[0434] The binding affinity of scTCR-Fab molecules TPP-70 to TPP-74 to HLA-A*02 monomers containing the PRAME-004 target peptide was analyzed by biolayer interferometry. Measurements were performed on an Octet RED384 system using manufacturer-recommended settings. The assay was performed with a sensor offset of 3 mm and an acquisition rate of 5 Hz. The binding reaction rate was measured at 30°C and a shaking rate of 1000 rpm using PBS, 0.05% Tween-20, and 0.1% BSA as buffer. After loading His-tagged HLA-A*02 / PRAME-004 monomers onto an HIS1K biosensor, serial dilutions of scTCR-Fab molecules were analyzed. Data evaluation was performed using Octet Data Analysis HT Software. Strong binding affinity was indicated in the range of 4 nM to 12 nM (K). D The values ​​were determined for the scTCR-Fab molecule (Table 4). Furthermore, scTCR-Fab mutants were screened for binding to 14 similar peptides (SEQ ID NOs: 187, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, and 212). Screening with similar peptides was essentially performed using the biolayer interferometry method described above, by analyzing scTCR-Fab molecules at a high concentration of 1 μM to enable the detection of weak binding signals. None of the mature scTCR mutants bound to any of the tested similar peptides (Figure 6). scTCR from TPP-74 was used to generate bispecific molecules in alternative formats such as the TCER® format. [Example 3]

[0435] T-cell engaging receptor [TCER®] format [Example 3.1] Preparation and Characterization of Soluble scTCR in Bispecificity TCER® Format To construct the TCER® molecule, DNA sequences encoding VH and VL, as well as sequences encoding V-alpha and V-beta and their respective linkers, were obtained by gene synthesis from either hUCHT1 (Var17), a novel humanized version of the anti-CD3 antibody UCHT1; BMA031 (V36), a humanized antibody that binds to the TCR / CD3 complex; or the anti-CD3 antibody ID4. The obtained DNA sequences were cloned in-frame into an expression vector encoding the hinge region, CH2, and CH3 domains derived from human IgG1 [Accession#:P01857]. The CH2 and CH3 domains were manipulated to include various mutations (including the N297Q mutation) to inhibit binding to the Fc gamma receptor and complement, and to incorporate a knob-into-hole structure into the CH3 domain where the interchain disulfide bond is further stabilized. The TCER® molecule (Tables 6 and 18) was prepared, purified, and characterized as outlined in Example 2.

[0436] [Table 6]

[0437] The functionality of the TCER® molecule in killing HLA-A*02-positive tumor cell lines (e.g., Hs695T) that present the PRAME-004 target peptide on their cell surface was evaluated using an LDH release assay. In addition, HLA-A*02-positive but PRAME-004-negative tumor cell lines (e.g., T98G) were evaluated to characterize the nonspecific or off-target activity of TCER® variants. Tumor cell lines were co-cultured with healthy HLA-A*02-positive donor-derived PBMCs in a 1:10 ratio, increasing the TCER® concentration. TCER®-induced cytotoxicity was quantified after 48 hours of co-culture by measuring released LDH. The EC of the dose-response curve was also analyzed. 50The values ​​were calculated using nonlinear four-point curve fitting. Representative results for the three TCER® molecules (Tables 6 and 18) are shown in Figures 7 and 8. These results demonstrate that all three TCER® molecules, which utilize various recruiting antibody domains, are functional and induce T cell-mediated cytotoxicity in a manner strictly dependent on PRAME-004. [Example 3.2]

[0438] Slot I The TCER® molecule was constructed using the VH and VL domains derived from hUCHT1 (Var17) or BMA031 (V36), as well as the V-alpha and V-beta domains described above (Example 3.1). The TCER® molecule (Tables 7 and 18) was prepared, purified, and characterized as outlined in Example 2.

[0439] [Table 7]

[0440] The binding affinity of TCER(registered trademark) Slot I mutants TPP-106, TPP-108~TPP-129 to the target peptide-HLA complex (HLA-A*02 / PRAME-004) was analyzed by biolayer interferometry. Measurements were performed using the Octet RED384 system as described above. Strong binding affinity was measured in the range of 3nM~10nM. D The values ​​were determined (Table 8). These data show further affinity improvement effects of TCR mutations bA84D and aN114Y, but mutations bT115L / K, bL11E, bP46M, bQ48R, and aN20K appear not to affect binding affinity. Furthermore, the binding affinity was determined for three selected analogous peptides that function as potential off-target peptides related to HLA-A*02, and compared with the binding of the target peptide-HLA. D A window was calculated. The strongest TCER(registered trademark) binding to similar peptides was observed with GIMAP8-001, and K DThe window size ranged from 26x to 168x. K was greater than 25x. D The window already offers a good treatment window.

[0441] [Table 8] [Example 3.3]

[0442] Slot II Further TCER® molecules were constructed using VH and VL domains derived from BMA031 (V36) or ID4, as well as the V-alpha and V-beta domains described above (Example 3.1). The preparation, purification, and characterization of each TCER® molecule (Tables 9 and 18) were carried out as outlined in Example 2, thereby purifying all ID4-based molecules using a MAbSelect SuRE column (GE Lifesciences).

[0443] [Table 9]

[0444] The binding affinity of TCER(registered trademark) Slot II mutants TPP-207~TPP-222 and TPP-227~TPP-230 to the target peptide-HLA complex (HLA-A*02 / PRAME-004) was analyzed by biolayer interferometry. Measurements were performed using the Octet RED384 system described above. Strong binding affinity was indicated by a K in the range of 1nM~7nM. D Determined by value (Table 10). When compared with the BMA031(V36) recruiter (TPP-219~TPP-222 vs. TPP-211~TPP-214), the same TCR variant (i.e., the same V) was combined with the ID4 recruiter. A and V BRegarding this, a higher binding affinity was observed. As observed with the TCER® molecule in slot I (Example 3.2), the affinity-improving effect of TCR mutations bA84D and aN114Y could also be confirmed with the TCER® mutants generated in slot II, but no effect on affinity was found with the mutations bT115L / K, bP46M, bQ48R, and aN20K.

[0445] The TCR binding motif was evaluated for selected TCER® molecules. To determine the binding motif, affinity was measured for the target peptide-HLA complex (HLA-A*02 / PRAME-004) and for complexes with PRAME-004 variants possessing alanine substitutions at positions 1, 3, 4, 5, 6, 7, or 8 of the peptide. Affinity measurements were performed using the Octet RED384 or HTX system described above. If a reduction of 50% or less in binding affinity or signal (measured at the highest concentration analyzed) was detected for the alanine-substituted peptide variant, the PRAME-004 position was considered to be part of the TCR binding motif. All TCER® variants exhibited a broad binding motif recognizing at least four positions of the PRAME-004 peptide (Table 10).

[0446] [Table 10] [Example 3.4]

[0447] Slot IIa Based on the data obtained for the previous TCER® variant (Example 3.3), novel variants were generated in which the positions of selected TCR amino acids were systematically substituted, resulting in positive effects on protein properties or binding properties that could be detected in the previous experiments. The preparation, purification, and characterization of each TCER® molecule (Tables 11 and 18) were carried out as outlined in Example 3.3. A summary of productivity and stress stability data is provided in Table 11.

[0448] [Table 11]

[0449] The binding affinity of TCER(registered trademark) Slot IIa mutants TPP-235~-250, -252~-268, -270, -277, and -279 to the target peptide-HLA complex (HLA-A*02 / PRAME-004) was analyzed by biolayer interferometry. Measurements were performed using the Octet RED384 or HTX system described above. Strong binding affinity was indicated by a K in the range of 2nM~15nM. D The values ​​were identified (Table 12). At position bA84, based on amino acid substitutions, bA84D was found to be the most preferred substitution. At position aN114, alternative amino acid substitutions corresponding to aN114Y in affinity (e.g., A, H, I, and L) were found. Alternatives for bT115K / with equivalent affinity were identified, including R, A, I, and V. Introducing the mutant bA110S slightly reduced the affinity of each mutant.

[0450] The binding motif was evaluated for selected TCER® variants. To determine the binding motif, affinity was measured for the target peptide-HLA complex (HLA-A*02 / PRAME-004) and for complexes with PRAME-004 variants possessing an alanine substitution at peptide positions 1, 3, 4, 5, 6, 7, or 8, as described above. If a reduction of 50% or less in binding affinity or signal (measured at the highest concentration analyzed) was detected for an alanine-substituted peptide variant, the PRAME-004 position was considered to be part of the TCR binding motif. All TCER® variants tested showed a broad binding motif recognizing at least three peptide positions (Table 12).

[0451] In addition to the binding motif, the binding specificity of selected TCER® Slot II and IIa variants was further analyzed by biolayer interferometry regarding binding to a set of 16 similar peptides that potentially function as off-target peptides. Measurements were basically performed using the Octet HTX system described above. For analysis, peptide-HLA complexes containing the PRAME-004 target peptide, individual peptides from the set of similar peptides, or control peptides were loaded onto the HIS1K biosensor, and the binding of TCER® variants was analyzed at a high TCER® concentration of 1 μM. Using the reaction signal at the end of the 5-minute association phase, the relative binding signal of the similar peptides compared to the PRAME-004 target peptide to the selected TCER® variants was calculated (Table 13). Under these conditions, binding events at very low affinity (e.g., K for binding to the PRAME-004 peptide:MHC complex) may be explained as not significant. D In comparison, K D Even if the binding increases by 25 times, 30 times, 40 times, 50 times, 75 times, or 100 times or more, it will still be detected. Of the 16 similar peptides analyzed, 11 showed no binding to any of the selected TCER(registered trademark) variants. For 5 of the 16 similar peptides, binding with a lower signal compared to PRAME-004 was detected, and 4 of these peptides were analyzed in more detail with respect to the TCER(registered trademark) Slot III variant (e.g., K compared to the PRAME-004 target peptide). D (The window was measured.)

[0452] [Table 12]

[0453] [Table 13] [Example 3.5]

[0454] Slot III As described above (Example 3.1), further TCER® molecules were constructed using the VH and VL domains, as well as V-alpha and V-beta, derived from BMA031 (V36) or its variants (A02 and D01), or ID4. Further TCER® molecules based on the UCHT1-V17 recruiting antibody (TPP-1109) were generated as references. DNA constructs encoding each molecule were generated as outlined above. The resulting plasmids were used for transfection of CHO-S cells by electroporation (MaxCyte) for transient expression and production of TCER® variants (Tables 14 and 18). Molecular purification, formulation, and initial characterization were carried out as outlined in Example 3.3 above.

[0455] [Table 14]

[0456] The ability of the TCER® molecule to kill HLA-A*02-positive tumor cell lines presenting various levels of PRAME-004 target peptides on the cell surface was evaluated using an LDH release assay. In addition, HLA-A*02-positive but PRAME-004-negative tumor cell lines (e.g., T98G) were evaluated to characterize the nonspecific or off-target activity of TCER® variants. Tumor cell lines were co-cultured with PBMC effectors derived from healthy HLA-A*02-positive donors in a 1:10 ratio, increasing the TCER® concentration. TCER®-induced cytotoxicity was quantified after 48 hours of co-culture by measuring released LDH. The EC of the dose-response curve was also analyzed. 50 The values ​​were calculated using nonlinear four-point curve fitting. EC for two PRAME-004-positive tumor cell lines (Hs695T and U20S) and a PRAME-004-negative tumor cell line (T98G). 50 The values ​​were determined in various experiments using different PBMC donors, and the overview is summarized in a graph in Figure 9.

[0457] The binding affinity of TCER(registered trademark) Slot III mutants TPP-214, -222, -230, -666, -669, -871, -872, -876, -879, -891, and -894 to the target peptide-HLA complex (HLA-A*02 / PRAME-004) was analyzed by biolayer interferometry. Measurements were performed at 30°C using an Octet HTX system. The assay was performed using a 16-channel HIS1K biosensor with a sensor offset of 3 mm and an acquisition rate of 5 Hz, with PBS, 0.05% Tween-20, and 0.1% BSA as the assay buffer. All binding affinities were measured by repeating the following assay process sequence: regeneration (5 seconds, 10 mM glycine pH 1.5) / neutralization (5 seconds, assay buffer; one regeneration cycle consists of four regeneration / neutralization repeats), baseline (60 seconds, assay buffer), loading (120 seconds, 10 μg / ml peptide-HLA), baseline (120 seconds, assay buffer), association (300 seconds, 2-fold serial dilutions of TCER® in the range of 100 nM to 1.56 nM or 50 nM to 0.78 nM, assay buffer as reference), dissociation (300 seconds, assay buffer). Data evaluation was performed using Octet Data Analysis HT Software. Subtraction of the reference sensor was performed to subtract the potential dissociation of peptide-HLA loaded onto the biosensor (via the biosensor loaded with peptide-HLA measured in buffer). Data tracking was aligned to a baseline (average of the last 5 seconds), inter-process corrections were applied to the dissociation process, Savitzky-Golay filtering was applied, and the curve was fitted overall using a 1:1 coupling model (Rmax is not linked by the sensor). Strong binding affinity was defined as K in the range of 2nM to 5nM. D The values ​​were determined (Table 15). Furthermore, the binding affinity was determined for four already identified potential off-target peptides and compared with the binding of the target peptide-HLA to K DThe window was calculated. Measurements were performed at 30°C using an Octet RED384 or HTX system. The assay was performed using a 16-channel mode HIS1K biosensor with a sensor offset of 3 mm and an acquisition rate of 5 Hz, with PBS, 0.05% Tween-20, and 0.1% BSA as assay buffers. All binding affinities were measured by repeating the following assay process sequence: regeneration (5 sec, 10 mM glycine pH 1.5) / neutralization (5 sec, assay buffer; one regeneration cycle consists of four regeneration / neutralization repeats), baseline (60 sec, assay buffer), loading (120 sec, 10 μg / ml peptide-HLA), baseline (120 sec, assay buffer), association (300 sec, 2-fold serial dilutions of TCER® in the range of 500 nM to 7.81 nM, assay buffer as reference), dissociation (300 sec, assay buffer). Data evaluation was performed using Octet Data Analysis HT Software. A reference sensor subtraction was performed to subtract the potential dissociation of peptide-HLA loaded onto the biosensor (through the biosensor loaded with each peptide-HLA measured in buffer). Data tracking was aligned to baseline (average of the last 5 seconds), inter-process corrections were made for the dissociation step, Savitzky-Golay filtering was applied, and the curves were fitted overall using a 1:1 binding model (Rmax is not linked by the sensor). Overall, fairly weak binding to potential off-target peptides compared to the target peptide was found in all variants, showing a window of at least 60x to no binding at all. NOMAP-3-1408 showed a relative binding signal equivalent to VIM-009, but K D It was not selected for the decision (Table 13). Regarding VIM-009, the smallest K measured was D The window was over 100 times (Table 15). Therefore, binding to VIM-009 was irrelevant, and affinity determination of NOMAP-3-1408 binding was considered unnecessary based on a binding signal equivalent to that of VIM-009. For one interaction, the K was 50 times. DA window was calculated. However, for this interaction and several other interactions, the Rmax values ​​calculated by the fitting algorithm were excessively low, suggesting that the interactions are weaker than calculated, and therefore the window is larger. Each interaction is shown in Table 15. To further analyze the specificity of different mutants, the binding motif was determined by measuring the affinity of the target peptide-HLA complex and alanine-substituted mutants to positions 1, 3, 4, 5, 6, 7, and 8. Measurements were performed at 30°C using an Octet HTX system. The assay was performed using a 16 or 8-channel HIS1K biosensor with a sensor offset of 3 mm and an acquisition rate of 5 Hz, using PBS, 0.05% Tween-20, and 0.1% BSA as assay buffer. All binding affinities were measured by repeating the following assay process sequence: regeneration (5 seconds, 10 mM glycine pH 1.5) / neutralization (5 seconds, assay buffer; one regeneration cycle consists of four regeneration / neutralization repeats), baseline (60 seconds, assay buffer), loading (120 seconds, 10 μg / ml peptide-HLA), baseline (120 seconds, assay buffer), association (150 seconds, 2-fold serial dilutions of TCER® ranging from 400 nM to 6.25 nM, assay buffer as reference), dissociation (300 seconds, assay buffer). Data evaluation was performed using Octet Data Analysis HT Software. Subtraction of the reference sensor was performed to subtract the potential dissociation of peptide-HLA loaded onto the biosensor (through the biosensor loaded with each peptide-HLA measured in buffer). Data tracking was aligned to a baseline (average of the last 5 seconds), inter-process corrections were applied to the dissociation process, Savitzky-Golay filtering was applied, and the curve was fitted overall using a 1:1 binding model (Rmax was not linked by the sensor). A location was considered part of the binding motif if the affinity or binding signal decreased to less than half (measured at the highest concentration analyzed).All TCER® variants tested exhibited broad binding motifs that recognized at least four and up to all analyzed peptide positions (Table 16). Positive effects on the binding motif were observed for bA84, aN114L, and bA110S / bT115A, which is consistent with previous data. For comparison, the binding motif of the alternative PRAME-004 targeted TCER® reference molecule (TPP-1109) was analyzed. This TCER® recognizes positions 5–8 of the peptide, and therefore binding is limited to this peptide stretch, whereas the positions recognized by the TCER® Slot III variant are more uniformly distributed throughout the peptide.

[0458] The TCER® slot III mutants TPP-214, -222, -230, -666, -669, -871, -872, -876, -879, -891, and -894 were further characterized in terms of their ability to kill T2 cells loaded with various levels of target peptides. After loading T2 cells with each concentration of PRAME-004 for 2 hours, the peptide-loaded T2 cells were co-cultured with human PBMCs in a 5:1 E:T ratio, increasing the concentration of the TCER® mutant over 48 hours. The level of LDH released into the supernatant was quantified using the CytoTox 96 Non-Radioactive Cytotoxicity Assay Kit (Promega). All TCER® mutants killed subpicomolar EC2 cells at a peptide loading concentration of 10 nM. 50 It showed potent killing of PRAME-004-loaded T2 cells at the specified values ​​(Figure 10, Table 17). EC 50 The values ​​increased with decreasing PRAME-004 loading levels. However, even at very low PRAME-004 loading concentrations of 10 pM, killing was induced by all TCER(registered trademark) variants except TPP-214.

[0459] [Table 15]

[0460] [Table 16]

[0461] [Table 17]

[0462] [Table 18]

[0463] In Table 18, with the exception of TPP-70, TPP-71, TPP-72, TPP-73, and TPP-74, the term "α-chain" is used in V α (That is, it refers to a polypeptide chain containing a variable domain derived from the TCRα chain.) The term "β chain" is V β This refers to a polypeptide chain containing a variable domain derived from the TCRβ chain. In the case of TPP-70, TPP-71, TPP-72, TPP-73, and TPP-74, the "α chain" does not contain any variable domain derived from any TCR, but the "β chain" contains two variable domains derived from TCRs (one from the TCRα chain and one from the TCRβ chain). [Example 3.6]

[0464] Safety evaluation of selected TCER(registered trademark) Slot III candidates The safety profiles (Tables 14-18) of TCER® molecules TPP-230, TPP-666, TPP-871, and TPP-891 were evaluated in cell killing experiments using astrocytes and cardiomyocytes (derived from induced pluripotent stem cells), as well as aortic endothelial cells, mesenchymal stem cells, and tracheal smooth muscle cells. Figure 11 shows the results of co-culture of the above normal cell types (all expressing HLA-A*02) with PBMC effector cells derived from healthy HLA-A*02+ donors at a ratio of 1:10 (target cells:effector cells) while increasing the TCER® concentration. Cells were co-cultured in a 1:1 mixture of normal tissue cell medium and T cell medium, or in T cell medium alone (LDH-AM). After 48 hours of co-culture, the supernatant was collected and LDH release was measured using LDH-Glo(trademark) Kit (Promega) to evaluate TCER(registered trademark)-induced normal tissue cell lysis. To determine the safety window, the TCER(registered trademark) molecule was co-cultured with the PRAME-004-positive tumor cell line Hs695T in a 1:1 mixture of normal tissue cell medium and T cell medium under the same conditions, and LDH release was subsequently evaluated.

[0465] As shown in Figure 11, TPP-230 and TPP-871 did not exhibit cytotoxicity to normal tissue cells even at the highest TCER® concentration of 100 nM. For TPP-666 and TPP-891, some degree of normal tissue cell lysis was observed at a 100 nM TCER® concentration, but no lysis was detected at 10 nM. Compared to Hs695T tumor cells, which showed significant lysis at 100 pM for all tested TCER® molecules and even at 10 pM for some molecules, lysis of normal tissue cells at a 100 nM concentration represents a safety window of 1,000 times (TPP-666 and TPP-891) or larger (TPP-230 and TPP-871). [Example 3.7]

[0466] Slot...

Claims

1. An antigen-binding protein that specifically binds to a PRAME antigen peptide containing or comprising the amino acid sequence SLLQHLIGL of SEQ ID NO: 50 and existing in complex with a major histocompatibility complex (MHC) protein, (a) A first polypeptide comprising the amino acid sequence of SEQ ID NO: 132 or a variable domain V A consisting of said amino acid sequence, and (b) A second polypeptide comprising the amino acid sequence of SEQ ID NO: 135 or a variable domain V B consisting of said amino acid sequence. Antigen-binding proteins that include this protein.

2. The antigen-binding protein according to claim 1, wherein the antigen-binding protein is a bispecific TCR antibody molecule.

3. The antigen-binding protein according to claim 1, wherein the first and second polypeptides are contained in two polypeptide chains.

4. Antibody light chain variable domain (V Ｌ ) and antibody heavy chain variable domain (V Ｈ ) further includes V L and V Ｈ CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD11b, CD11c, CD14, CD16, CD18, CD22, CD25 , CD28, CD32a, CD32b, CD33, CD41, CD41b, CD42a, CD42b, CD44, CD45RA, CD49, CD55 , CD56, CD61, CD64, CD68, CD90, CD94, CD95, CD117, CD123, CD125, CD134, CD137, C D152, CD163, CD193, CD203c, CD235a, CD278, CD279, CD287, Nkp46, NKG2D, GITR, F c. It binds to antigens selected from the group consisting of εRI, TCRα / β and TCRγ / δ, HLA-DR, and 4-1 BB, or combinations thereof, and / or binds to effector cells. The antigen-binding protein according to claim 1.

5. The antigen-binding protein comprises a first and a second polypeptide chain. The first polypeptide chain is given by formula [Ia]: V １ -L １ -D １ -L ２ -V ２ -L ３ -D ２ [Ia] It is represented by, The second polypeptide chain is of formula [IIa] 6 ３ -8 ４ -0 ３ -8 ５ -6 ４ -8 ６ -0 ４ [HEY] It is represented by, During the ceremony, - V １ , V ２ , V ３ , and V ４ is a variable domain, V １ ~V ４ One of them is V Ａ And one is, V Ｂ And one is V Ｌ And one of them is V Ｈ And; - D １ , D ２ , D ３ , and D ４ This is a dimerization domain, and may or may not be present, D １ and D ３ , and D ２ and D ４ They bind to each other specifically, D １ and D ３ or D ２ and D ４ At least one pair of these exists, - L １ , L ２ , L ３ , L ４ , L ５ , and L ６ It is a linker, L １ and L ４ It exists, L ２ , L ３ , L ５ , and L ６ It may or may not exist. The antigen-binding protein according to claim 4.

6. The antigen-binding protein comprises a first and a second polypeptide chain, the first polypeptide chain having formula [Ib]: V Ｌ -L １ -V Ｂ -F ｃ１ [Ib] It is represented as, The second polypeptide chain is given by formula [IIb]: V Ａ -L ４ -V Ｈ -F ｃ２ [IIb] It is represented as, During the ceremony, - V Ａ It consists of the amino acid sequence of sequence number 132, V Ｂ This consists of the amino acid sequence of sequence number 135; - L １ and L ４ It is a linker; - V Ｌ and V Ｈ These are antibody variable domains that, together, form an antigen-binding site that binds to CD3 or the TCRα / βCD3 complex; - F ｃ１ and F ｃ２ These are a pair of FC domains, The antigen-binding protein according to claim 5.

7. The antigen-binding protein according to claim 6, wherein F c1 consists of the amino acid sequence of SEQ ID NO: 149 (nob) and F c2 consists of the amino acid sequence of SEQ ID NO: 150 (whole).

8. - A first polypeptide chain that is at least 90% identical to Sequence ID No. 158, and - A second polypeptide chain that is at least 90% identical to Sequence ID No.

300. The antigen-binding protein according to claim 1, comprising:

9. The antigen-binding protein according to claim 1, comprising a first polypeptide chain described in SEQ ID NO: 158 and a second polypeptide chain described in SEQ ID NO:

300.

10. An isolated nucleic acid comprising a sequence encoding an antigen-binding protein as described in claim 1.

11. A host cell comprising an antigen-binding protein according to any one of claims 1 to 9, or a nucleic acid according to claim 10.

12. A pharmaceutical composition comprising an antigen-binding protein according to any one of claims 1 to 9 or a nucleic acid according to claim 10, and a pharmaceutically acceptable carrier.

13. A pharmaceutical composition comprising the host cell described in claim 11 and a pharmaceutically acceptable carrier.

14. The pharmaceutical composition according to claim 12, wherein the antigen-binding protein is dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.

15. A method for producing the antigen-binding protein described in claim 1, a. Prepare the host cells. b. Preparing a gene construct comprising a coding sequence encoding the antigen-binding protein described in claim 1. c. Introducing the gene construct into the host cell, and d. Expressing the gene construct in the host cells. A method that includes this.

16. An antigen-binding protein according to any one of claims 1 to 9, or a nucleic acid according to claim 10, for use in pharmaceuticals.

17. An antigen-binding protein according to any one of claims 1 to 9, or a nucleic acid according to claim 10, for use in the diagnosis, prevention, and / or treatment of cancer, wherein the cancer is selected from the group of cancers consisting of acute myeloid leukemia, breast cancer, cholangiocarcinoma, gallbladder cancer, glioblastoma, hepatocellular carcinoma, squamous cell carcinoma of the head and neck, melanoma, melanoma without pigment, non-Hodgkin lymphoma, non-small cell lung cancer adenocarcinoma, non-small cell lung cancer, squamous non-small cell lung cancer, ovarian cancer, esophageal cancer, renal cell carcinoma, small cell lung cancer, bladder cancer, uterine and endometrial cancer, osteosarcoma, chronic lymphocytic leukemia, colorectal cancer, and synovial sarcoma.

18. Use of an antigen-binding protein according to any one of claims 1 to 9 or a nucleic acid according to claim 10 in the manufacture of a pharmaceutical product for the diagnosis, prevention, and / or treatment of cancer.

19. The use according to claim 18, wherein the cancer is selected from the group of cancers consisting of acute myeloid leukemia, breast cancer, cholangiocarcinoma, gallbladder cancer, glioblastoma, hepatocellular carcinoma, squamous cell carcinoma of the head and neck, melanoma, melanoma without pigmentation, non-Hodgkin lymphoma, adenocarcinoma of non-small cell lung cancer, non-small cell lung cancer, squamous cell non-small cell lung cancer, ovarian cancer, esophageal cancer, renal cell carcinoma, small cell lung cancer, bladder cancer, uterine and endometrial cancer, osteosarcoma, chronic lymphocytic leukemia, colorectal cancer, and synovial sarcoma.

Images