CN114144429B - Bispecific antibodies against PD-1 and LAG-3 - Google Patents

Abstract

The present invention provides bispecific antibodies comprising: a first polypeptide chain comprising, from N-terminus to C-terminus, a first heavy chain Variable (VH) domain of a PD-1 antibody, a first T Cell Receptor (TCR) constant region operably linked, and a second VH domain of a LAG-3 antibody; a second polypeptide chain comprising, from N-terminus to C-terminus, a first light chain Variable (VL) domain of a PD-1 antibody and an operably linked second TCR constant region; a third polypeptide chain comprising, from N-terminus to C-terminus, a second VL domain of a LAG-3 antibody and an operably linked antibody light chain Constant (CL) domain. The invention also provides amino acid sequences of the antibodies of the invention, cloning or expression vectors, host cells and methods for expressing or isolating the antibodies, therapeutic compositions comprising the antibodies of the invention, and methods of treating cancer and other diseases using the bispecific antibodies.

Description

Bispecific antibodies against PD-1 and LAG-3

Technical Field

The present invention relates to bispecific antibodies. Furthermore, the invention provides polynucleotides encoding the antibodies, vectors comprising the polynucleotides, host cells, methods of producing the antibodies, and immunotherapies using the bispecific antibodies in the treatment of cancer, infection, or other human diseases.

Background

Over the past few years, immunotherapy has evolved into a very promising new front against certain cancer types. PD-1 as one of the immune checkpoint proteins is in activated CD4 ⁺ T cells and CD8 ⁺ An inhibitory member of the CD28 family expressed on T cells and B cells. PD-1 plays a major role in down-regulating the immune system.

PD-1 is a type I transmembrane protein whose structure consists of an immunoglobulin variable region-like extracellular domain and a cytoplasmic domain containing an Immunoreceptor Tyrosine Inhibitory Motif (ITIM) and immunoreceptor tyrosine opening Guan Jixu (ITSM).

PD-1 has two known ligands, PD-L1 and PD-L2, which are members of the B7 family of cell surface expression. After ligation to its physiological ligand, PD-1 inhibits T-cell activation by recruiting SHP-2, which dephosphorylates and inactivates Zap70, a major integrator of T-cell receptor (TCR) mediated signaling. As a result, PD-1 inhibits T cell proliferation and T cell functions such as cytokine production and cytotoxic activity.

Monoclonal antibodies targeting PD-1 can block PD-1/PD-L1 binding and enhance immune responses against cancer cells. These drugs have shown great promise in the treatment of certain cancers. Several pharmaceutical companies have developed a number of therapeutic antibodies targeting PD-1 in batches including pembrolizumab (Keytruda), nivolumab (Opdivo), cemiplimab (Libtayo). These drugs have been shown to be effective in treating a variety of different types of cancers including cutaneous melanoma, non-small cell lung cancer, renal cancer, bladder cancer, head and neck cancer, and hodgkin's lymphoma. They are also being studied for use against many other types of cancer.

Lymphocyte activation gene 3, also known as LAG-3, is a type I transmembrane protein, a member of the immunoglobulin superfamily (IgSF). LAG-3 is a cell surface molecule expressed on activated T cells, NK cells, B cells, and plasmacytoid dendritic cells but not on resting T cells. LAG-3 shares approximately 20% amino acid sequence homology with CD4, but binds to MHC class II with higher affinity, providing negative regulation of T cell receptor signaling.

Blocking LAG-3 in vitro enhances T cell proliferation and cytokine production, and LAG-3 deficient mice have a defect in down-regulation of T cell responses induced by superantigen staphylococcal enterotoxin B, peptide or sendai virus infection. LAG-3 is found in activated natural Treg (nTreg) and induced CD4 ⁺ FoxP3 ⁺ Treg (iTreg) cells, in which the expression level is higher than in activated effector CD4 ⁺ Levels observed on T cells. Blocking LAG-3 on Treg cells abrogates Treg cell inhibition function, whereas LAG-3 on non-Treg CD4 ⁺ Ectopic expression in T cells confers inhibitory activity. Based on the immunomodulatory effects of LAG-3 on T cell function in chronic infection and cancer, the predictive mechanism of action of LAG-3 specific monoclonal antibodies is to suppress the negative regulation of tumor-specific effector T cells. Furthermore, in mice and humans, dual blockade of the PD-1 pathway and LAG-3 has been shown to be more effective than blocking either molecule alone against tumor immunity.

In antigen-specific CD8 ⁺ Co-expression of LAG-3 and PD-1 was found on T cells and co-blocking of both resulted in increased proliferation and cytokine production. The combination of anti-LAG-3 therapy with anti-PD-1 therapy has entered clinical trials for a variety of different types of solid tumors.

Disclosure of Invention

The present invention provides isolated antibodies, particularly bispecific antibodies.

In one aspect, the invention provides a bispecific antibody or antigen binding fragment thereof comprising:

a first polypeptide chain comprising, from N-terminus to C-terminus, a first heavy chain Variable (VH) domain of a PD-1 antibody, a first T Cell Receptor (TCR) constant region operably linked, and a second VH domain of a LAG-3 antibody,

a second polypeptide chain comprising, from N-terminus to C-terminus, a first light chain Variable (VL) domain of a PD-1 antibody and an operably linked second TCR constant region,

a third polypeptide chain comprising, from N-terminus to C-terminus, a second VL domain of a LAG-3 antibody and an operably linked antibody light chain Constant (CL) domain,

wherein the first TCR constant region and the second TCR constant region are capable of forming dimers comprising at least one unnatural inter-chain disulfide bond,

wherein the second VH domain of the LAG-3 antibody comprises H-CDR1, H-CDR2, and H-CDR3; wherein the H-CDR3 comprises the amino acid sequence as set forth in SEQ ID NO:1 and conservative modifications thereof; the H-CDR2 comprises the amino acid sequence as set forth in SEQ ID NO:2 and conservative modifications thereof; the H-CDR1 comprises the amino acid sequence as set forth in SEQ ID NO:3 and conservative modifications thereof,

Wherein the first VH domain of the PD-1 antibody comprises H-CDR1, H-CDR2, H-CDR3; wherein the H-CDR3 comprises the amino acid sequence as set forth in SEQ ID NO:4 and conservative modifications thereof; the H-CDR2 comprises the amino acid sequence as set forth in SEQ ID NO:5 and conservative modifications thereof; the H-CDR1 comprises the amino acid sequence as set forth in SEQ ID NO:6 and conservative modifications thereof.

In one embodiment, the invention provides an antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof provides a bivalent binding site specific for PD-1 or LAG-3.

In one embodiment, the invention provides an antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises two of said first polypeptide chains, two of said second polypeptide chains and two of said third polypeptide chains.

In one embodiment, the invention provides an antibody or antigen-binding fragment thereof, the second VL domain of which comprises L-CDR1, L-CDR2 and L-CDR3; wherein said L-CDR3 comprises the amino acid sequence as set forth in SEQ ID NO:7 and conservative modifications thereof; the L-CDR2 comprises the amino acid sequence as set forth in SEQ ID NO:8 and conservative modifications thereof; the L-CDR1 comprises the amino acid sequence as set forth in SEQ ID NO:9 and conservative modifications thereof.

In one embodiment, the invention provides an antibody or antigen-binding fragment thereof, the second VL domain of which comprises L-CDR1, L-CDR2 and L-CDR3; wherein said L-CDR3 comprises the amino acid sequence as set forth in SEQ ID NO:10 and conservative modifications thereof; the L-CDR2 comprises the amino acid sequence as set forth in SEQ ID NO:11 and conservative modifications thereof; the L-CDR1 comprises the amino acid sequence as set forth in SEQ ID NO:12 and conservative modifications thereof.

In one embodiment, the invention provides an antibody or antigen-binding fragment thereof, wherein the second VH domain of the LAG-3 antibody comprises an amino acid sequence that hybridizes to SEQ ID NO:13, at least 70%, 80%, 85%, 90%, 95% or 99% homologous sequence.

In one embodiment, the invention provides an antibody or antigen-binding fragment thereof, wherein the first VH domain of the PD-1 antibody comprises an amino acid sequence that hybridizes to SEQ ID NO:14, at least 70%, 80%, 85%, 90%, 95% or 99% homologous sequence.

In one embodiment, the invention provides an antibody or antigen-binding fragment thereof, wherein the second VL domain of the LAG-3 antibody comprises an amino acid sequence that hybridizes to SEQ ID NO:15, at least 70%, 80%, 85%, 90%, 95% or 99% homologous sequence.

In one embodiment, the invention provides an antibody or antigen-binding fragment thereof, wherein the first VL domain of the PD-1 antibody comprises an amino acid sequence that hybridizes to SEQ ID NO:16 at least 70%, 80%, 85%, 90%, 95% or 99% homologous sequence.

In one embodiment, the invention provides an antibody or antigen-binding fragment thereof, wherein the second VH domain of the LAG-3 antibody comprises SEQ ID NO: 13.

In one embodiment, the invention provides an antibody or antigen-binding fragment thereof, wherein the first VH domain of the PD-1 antibody comprises SEQ ID NO: 14.

In one embodiment, the invention provides an antibody or antigen-binding fragment thereof, wherein the second VL domain of the LAG-3 antibody comprises SEQ ID NO: 15.

In one embodiment, the invention provides an antibody or antigen-binding fragment thereof, wherein the first VL domain of the PD-1 antibody comprises the amino acid sequence of SEQ ID NO: 16.

In one embodiment, the invention provides an antibody or antigen binding fragment thereof, wherein the first TCR constant region comprises an engineered TCR β constant region comprising one or more mutant residues that replace wild-type amino acid residues in the TCR β constant region. The mutated residues in the TCR β constant region are selected from K9E, S56C, N69Q and C74A. The second TCR constant region comprises an engineered TCR alpha constant region comprising one or more mutated residues replacing a wild-type amino acid residue in the TCR alpha constant region. The mutant residues in the TCR α constant region are selected from N32Q, T47C, N Q and N77Q.

In one embodiment, the engineered TCR β constant region comprises SEQ ID NO:21, said engineered TCR α constant region comprising the sequence of SEQ ID NO: 22.

In one embodiment, the invention provides an antibody or antigen-binding fragment thereof, the first TCR constant region and the second VH domain are encoded by SEQ ID NO:23, and a peptide sequence of 23.

In one embodiment, the invention provides an antibody or antigen binding fragment thereof, the first polypeptide further comprising an antibody heavy chain constant CH1 domain.

In one embodiment, the first polypeptide further comprises an IgG Fc fragment, wherein the IgG Fc fragment is operably linked to the CH1 domain.

In one embodiment, the first polypeptide comprises SEQ ID NO:17 or 20.

In one embodiment, the second polypeptide comprises SEQ ID NO: 18.

In one embodiment, the third polypeptide comprises SEQ ID NO: 19.

The above antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment

a) At a K of 9.88E-10 or less _D Binds to human PD-1; and is also provided with

b) At a K of 1.70E-11 or less _D Binds to human LAG-3.

The sequences of the antibodies are shown in table 1 and the sequence listing.

TABLE 1 deduced amino acid sequence of antibodies

The CDR sequences of the antibodies are shown in table 2 and the sequence listing.

TABLE 2 CDR sequences of antibodies

The antibodies of the invention may be humanized or fully human.

In another aspect, the invention provides a nucleic acid molecule encoding the or an antigen binding fragment thereof.

The present invention provides a cloning or expression vector comprising a nucleic acid molecule encoding said antibody or antigen binding fragment thereof.

The invention also provides a host cell comprising one or more cloning or expression vectors.

In another aspect, the invention provides a method comprising culturing a host cell of the invention and isolating the antibody.

In another aspect, the invention provides a pharmaceutical composition comprising an antibody or antigen-binding fragment of the antibody of the invention and one or more pharmaceutically acceptable excipients, diluents or carriers.

The present invention provides an immunoconjugate comprising an antibody or antigen-binding fragment thereof of the invention linked to a therapeutic agent.

Wherein the invention provides a pharmaceutical composition comprising said immunoconjugate and one or more pharmaceutically acceptable excipients, diluents or carriers.

The invention also provides a method of modulating an immune response in a subject, the method comprising administering to the subject an antibody or antigen-binding fragment of any of the antibodies of the invention.

The invention also provides the use of the antibody or antigen binding fragment thereof for the manufacture of a medicament for the treatment or prophylaxis of immune disorders or cancer.

The invention also provides a method of inhibiting the growth of a tumor cell in a subject, the method comprising administering to the subject a therapeutically effective amount of the antibody or the antigen-binding fragment to inhibit the growth of the tumor cell.

Wherein the invention provides the method wherein the tumor cell is a cell of a cancer selected from the group consisting of melanoma, renal cancer, prostate cancer, breast cancer, colon cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer and rectal cancer.

Features and advantages of the invention

Bispecific antibodies directed against both the PD-1 and LAG-3 pathways may provide several benefits in cancer therapy. The bispecific antibodies can increase the response rate to PD-1 and LAG-3 biscationic cancers compared to anti-PD-1 therapies.

Drawings

FIG. 1 shows binding of PD-1 XLAG-3 bispecific antibodies to cell surface human PD-1.

FIG. 2PD-1 XLAG-3 bispecific antibody binds to cell surface human LAG-3.

FIG. 3PD-1 XLAG-3 bispecific antibody binds to cell surface cynomolgus monkey PD-1.

FIG. 4PD-1 XLAG-3 bispecific antibody binds to cell surface cynomolgus monkey LAG-3.

FIG. 5 shows the binding of PD-1 XLAG-3 bispecific antibodies to mouse PD-1 and LAG-3. FIG. 5A shows that PD-1 XLAG-3 bispecific antibody does not bind to mouse PD-1 and FIG. 5B shows that PD-1 XLAG-3 bispecific antibody does not bind to mouse LAG-3.

FIG. 6 shows binding of PD-1 XLAG-3 bispecific antibodies to human CD4, CTLA-4 and CD28 proteins. FIG. 6A shows that PD-1 XLAG-3 bispecific antibody does not bind to human CTLA-4 protein, FIG. 6B shows that PD-1 XLAG-3 bispecific antibody does not bind to human CD28 protein, and FIG. 6C shows that PD-1 XLAG-3 bispecific antibody does not bind to human CD4 protein.

FIG. 7 shows binding of PD-1 XLAG-3 bispecific antibodies to human PD-1 and LAG-3 proteins.

FIG. 8 shows that PD-1 XLAG-3 bispecific antibodies block the binding of PD-1 to cells expressing PD-L1.

FIG. 9 shows that PD-1 XLAG-3 bispecific antibodies block binding of LAG-3 to MHC-II.

FIG. 10 shows that PD-1 XLAG-3 bispecific antibodies enhance the NFAT pathway in Jurkat expressing PD-1 and LAG-3.

FIG. 11 shows the effect of PD-1 XLAG-3 bispecific antibodies on human allogeneic Mixed Lymphocyte Reaction (MLR). FIG. 11A shows that PD-1 XLAG-3 bispecific antibodies enhanced IL-2 production in the MLR assay and FIG. 11B shows that PD-1 XLAG-3 bispecific antibodies enhanced IFN-gamma production in the MLR assay.

Fig. 12 shows tumor volume and survival curves of treated mice. FIG. 12A shows that PD-1 XLAG-3 bispecific antibodies inhibited B16F10 tumor growth in transgenic mice and FIG. 12B shows the body weight of treated mice.

FIG. 13 shows a schematic representation of PD-1 XLAG-3 bispecific antibody configuration. The antibody comprises two sets of three polypeptide chains, each set comprising: 1) VL (PD-1) -engineered tcra constant region; 2) VL (LAG-3) -CL; and 3) VH (PD-1) -engineered TCR β constant region-VH (LAG-3) -CH 1-hinge-CH 2-CH3.

Detailed Description

The following description of the present disclosure is intended only to illustrate various embodiments of the present disclosure. Therefore, the specific modifications discussed should not be construed as limiting the scope of the disclosure. It will be apparent to those skilled in the art that various equivalents, changes, and modifications can be made without departing from the scope of the disclosure, and it is to be understood that such equivalent embodiments are to be included herein. All references, including publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

References to "a", "an", and "the" are used herein to refer to one or more than one (i.e., at least one) of the named objects. For example, "polypeptide complex" means one polypeptide complex or more than one polypeptide complex.

As used herein, the term "about" or "approximately" refers to a quantity, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length that varies by up to 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% relative to a reference quantity, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length. In particular embodiments, the term "about" or "approximately" when preceded by a numerical value means that the value is plus or minus 15%, 10%, 5%, or 1% of the range.

Throughout this disclosure, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. "consisting of … …" means any item including, but not limited to, following the phrase "consisting of … …". Thus, the phrase "consisting of … …" means that the listed elements are required or mandatory and that no other elements may be present. "consisting essentially of … …" is meant to include any element listed after the phrase and is limited to other elements that do not interfere with or contribute to the activity or effect specified for the listed elements in this disclosure. Thus, the phrase "consisting essentially of … …" means that the listed elements are necessary or mandatory, but other elements are optional and may or may not be present depending on whether they affect the activity or function of the listed elements.

The phrases "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues or a collection of polymers of multiple amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acid, as well as naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimics that function in a similar manner to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are modified at a later time, such as hydroxyproline, gamma-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs refer to analogs having the same basic chemical structure as a naturally occurring amino acid, i.e., an alpha-carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. These analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. The α -carbon refers to the first carbon atom attached to a functional group such as a carbonyl group. Beta carbon refers to the second carbon atom attached to the alpha carbon, and the system continues to name the carbons alphabetically with the greek alphabet amine. Amino acid mimetics refers to chemical compounds that have a structure that differs from the general chemical structure of an amino acid, but that function in a manner similar to a naturally occurring amino acid. The term "protein" generally refers to a large polypeptide. The term "peptide" generally refers to a short polypeptide. Polypeptide sequences are generally described as having an amino terminus (N-terminus) at the left end of the polypeptide sequence and a carboxy terminus (C-terminus) at the right end of the polypeptide sequence. As used herein, a "polypeptide complex" refers to a complex comprising one or more polypeptides that are combined to perform certain functions. In certain embodiments, the polypeptide is immune-related.

As referred to herein, the term "antibody" includes whole antibodies and any antigen-binding fragment (i.e., an "antigen-binding portion") or single chain thereof. "antibody" refers to a protein or antigen binding portion thereof comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region comprises one domain CL. The VH and VL regions may be further subdivided into regions of highly variable nature known as Complementarity Determining Regions (CDRs) interspersed with regions that are more conserved known as Framework Regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. CDRs in the heavy chain are abbreviated as H-CDRs, e.g., H-CDR1, H-CDR2, H-CDR3, and CDRs in the light chain are abbreviated as L-CDRs, e.g., L-CDR1, L-CDR2, L-CDR3.

The term "antibody" as used in this disclosure refers to an immunoglobulin or fragment or derivative thereof, and encompasses any polypeptide comprising an antigen binding site, whether produced in vitro or in vivo. The term includes, but is not limited to, polyclonal, monoclonal, monospecific, multispecific, non-specific, humanized, single-chain, chimeric, synthetic, recombinant, hybrid, mutant, and grafted antibodies. The term "antibody" also includes antibody fragments, such as scFv, dAb, bispecific antibodies comprising a first VH domain and a second VH domain, and other antibody fragments that retain the antigen-binding function, i.e. the ability to specifically bind PD-1 and LAG-3. Typically, such fragments will comprise antigen-binding fragments.

An antigen binding fragment typically comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH), however, it does not necessarily comprise both. For example, so-called Fd antibody fragments consist only of VH and CH1 domains, but still retain some of the antigen-binding function of the intact antibody.

For antibodies, "Fc" refers to a portion of the antibody that consists of the second (CH 2) and third (CH 3) constant regions of the first heavy chain that are bound to the second and third constant regions of the second heavy chain by disulfide bonds. The Fc portion of an antibody is responsible for a variety of different effector functions such as ADCC and CDC, but does not play a role in antigen binding.

As used herein, a "CH2 domain" refers to a portion of a heavy chain molecule that extends from, for example, about amino acid 244 to amino acid 360 of an IgG antibody using conventional numbering schemes (amino acids 244 to 360 of the Kabat numbering system, and amino acids 231-340 of the EU numbering system; see Kabat, e.g., U.S. part of Health and Human Services, (1983)).

The "CH3 domain" extends from the CH2 domain to the C-terminus of the IgG molecule and comprises about 108 amino acids. Certain immunoglobulin classes, such as IgM, also contain CH4 regions.

As used herein, the term "antigen binding moiety" refers to an antibody fragment formed from a portion of an antibody that comprises one or more CDRs, or any other antibody fragment that binds an antigen but does not comprise the complete native antibody structure.

The terms "programmed death protein 1", "programmed cell death protein 1", "protein PD-1", "PD1", "PDCD1", "hPD-1", "CD279" and "hPD-F" are used interchangeably and include variants, isotypes, species homologs of human PD-1, PD-1 of other species and analogs having at least one epitope in common with PD-1.

The terms "LAG-3", "lymphocyte activation gene 3", "CD223" are used interchangeably herein and include variants, isotypes, species homologs of human LAG-3, LAG-3 of other species and analogs having at least one epitope in common with LAG-3.

The term "cross-reactive" refers to the binding of an antigenic fragment described herein to the same target molecule in humans, monkeys and/or rats (mice or rats). Thus, "cross-reactivity" should be understood as inter-species reactivity with the same molecule X expressed in a different species, but not with molecules other than X. Monoclonal antibodies that recognize, for example, human PD-1, are species-specific across monkey and/or murine (mouse or rat) PD-1, as determined, for example, by FACS analysis.

The term "conservative modification" refers to nucleotide and amino acid sequence modifications that do not significantly affect or alter the binding characteristics of an antibody encoded by or containing the amino acid sequence. Such conservative sequence modifications include nucleotide and amino acid substitutions, additions, and deletions. Modifications may be introduced into the sequence by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

As used herein, the terms "homologue" and "homologous" are used interchangeably and refer to a nucleic acid sequence (or its complementary strand) or amino acid sequence having at least 70% (e.g., at least 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity when optimally aligned with another sequence.

For amino acid sequences (or nucleic acid sequences), "percent (%) sequence identity" is defined as the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to amino acid (or nucleic acid) residues in a reference sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum number of identical amino acids (or nucleic acids). Conservative substitutions of amino acid residues may or may not be considered as identical residues. Alignment for the purpose of determining percent amino acid (or Nucleic acid) sequence identity can be achieved, for example, using publicly available tools such as BLASTN, BLASTp (available on the website of the National Center for Biotechnology Information (NCBI), see also Altschul s.f. et al, j. Mol. Biol.,215:403-410 (1990), stephen f. Et al, nucleic Acids res.,25:3389-3402 (1997)), clustalW2 (available on the website of the european Bioinformatics institute, see also Higgins d.g. et al, methods in Enzymology,266:383-402 (1996), larkin m.a. et al, bioinformatics (Oxford, england), 23 (21): 2947-8 (2007)) and ALIGN or Megalign (DNASTAR) software. The default parameters provided by the tool may be used by those skilled in the art, or parameters suitable for alignment may be customized, for example, by selecting a suitable algorithm.

As used herein, the term "specific binding" or "specifically binding" refers to a non-random binding reaction between two molecules, e.g., between an antibody and an antigen. In certain embodiments, the polypeptide complexes and bispecific polypeptide complexes provided herein are provided at 10 or less ^-6 M (e.g.. Ltoreq.5X10) ^-7 M、≤2×10 ^-7 M、≤10 ^-7 M、≤5×10 ^-8 M、≤2×10 ^-8 M、≤10 ^-8 M、≤5×10 ^-9 M、≤2×10 ^-9 M、≤10 ^-9 M or less than or equal to 10 ^-10 M) binding affinity (K _D ) Specifically bind to the antigen. When used herein, K _D Refers to the ratio of dissociation rate to association rate (k off/k on), and may be used, for example, using an instrument such as Biacore, using surface plasmonThe subresonance method.

The term "operably linked" refers to the juxtaposition of two or more biological sequences of interest, with or without a spacer or linker, such that they are in a relationship permitting them to function in the intended manner. When used in reference to a polypeptide, it is intended to mean that the polypeptide sequences are linked in a manner that allows the ligation product to have the intended biological activity. For example, an antibody variable region may be operably linked to a constant region so as to provide a stable product with antigen binding activity. The term may also be used to refer to polynucleotides. For example, when a polynucleotide encoding a polypeptide is operably linked to a regulatory sequence (e.g., a promoter, enhancer, silencer sequence, etc.), it is intended to mean that the polynucleotide sequences are linked in a manner that allows for the regulated expression of the polypeptide from the polynucleotide.

As used herein, the term "mutation" or "mutated" with respect to an amino acid sequence refers to the substitution, insertion or addition of an amino acid residue.

Bispecific configuration

Provided herein is a novel bispecific antibody or antigen binding fragment thereof comprising: a first polypeptide chain comprising, from N-terminus to C-terminus, a first heavy chain variable VH domain of a PD-1 antibody operably linked to a first TCR constant region and a second VH domain of a LAG-3 antibody; a second polypeptide comprising, from N-terminus to C-terminus, a first VL domain of a PD-1 antibody operably linked to a second TCR constant region; a third polypeptide comprising a second VL domain of a LAG-3 antibody from N-terminus to C-terminus, wherein the first TCR constant region and the second TCR constant region are capable of forming a dimer comprising at least one interchain disulfide bond (figure 13).

In one aspect, the disclosure herein provides a bispecific polypeptide complex. As used herein, the term "bispecific" means that there are two antigen binding moieties, each of which is capable of specifically binding to a different antigen. Bispecific polypeptide complexes provided herein comprise a first antigen binding moiety comprising a first heavy chain variable domain operably linked to a first TCR constant region (tcrp) and a first light chain variable domain operably linked to a second TCR constant region (tcra), wherein the first TCR constant region and the second TCR constant region are capable of forming a dimer comprising at least one unnatural stabilizing interchain bond. The bispecific polypeptide complexes provided herein also comprise a second antigen binding moiety comprising a second antigen binding site but no sequence derived from a TCR constant region.

It has surprisingly been found that the polypeptide complexes provided herein with at least one non-natural interchain bond, particularly a non-natural disulfide bond, can be recombinantly expressed and assembled into a desired conformation, stabilizing the TCR constant region dimers while providing good antigen-binding activity of the antibody variable region. In addition, the polypeptide complexes were found to be well-tolerated by conventional antibody engineering, such as modification of glycosylation sites and removal of certain native sequences. Furthermore, the polypeptide complexes provided herein can be incorporated into bispecific configurations, which can be readily expressed and assembled with little or substantially no mismatches in antigen binding sequences due to the presence of TCR constant regions in the polypeptide complexes. Other advantages of the polypeptide complexes and constructs provided herein will become more apparent in the disclosure that follows.

In certain embodiments, the first and/or second antigen binding moiety is bivalent. The term "bivalent" means that there are two binding sites in the antigen binding molecule, respectively. In certain embodiments, this provides for greater binding to an antigen or epitope than the monovalent counterpart. In certain embodiments, in the divalent antigen binding moiety, the first valence of the binding site and the second valence of the binding site are structurally identical (i.e., have the same sequence).

TCR constant region

The human TCR alpha chain constant region is designated TRAC, NCBI accession number P01848 (https:// www.uniprot.org/uniprot/P01848), the sequence of the WT TCR alpha domain being: IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESS (SEQ ID NO: 26); the engineered TCR α chain constant regions of the invention comprise one or more mutation sites selected from the group consisting of N32Q, T47C, N Q and N77Q.

The human tcrp chain constant region has two different variants, known as TRBC1 and TRBC2 (IMGT nomenclature). In the present invention, the sequence of the wild-type TCR β domain is DLKNVFPPKVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGR (SEQ ID NO: 27), NCBI accession number A0A5B9 (https:// www.uniprot.org/uniprot/A0A5B 9), and the engineered TCR β domain comprises one or more mutation sites selected from K9E, S56C, N Q and C74A.

In the present disclosure, the first and second TCR constant regions of the polypeptide complexes provided herein are capable of forming dimers that comprise at least one non-native inter-chain bond between the TCR constant regions that is capable of stabilizing the dimers.

As used herein, the term "dimer" refers to an association structure formed by covalent or non-covalent interactions of two molecules, such as polypeptides or proteins. Homodimers or homodimers are formed from two identical molecules, heterodimers or heterodimers are formed from two different molecules. The dimer formed by the first and second TCR constant regions is a heterodimer.

An inter-chain bond is formed between one amino acid residue on one TCR constant region and another amino acid residue on another TCR constant region. In certain embodiments, the non-native inter-chain bond may be any bond or interaction capable of associating two TCR constant regions into a dimer. Examples of suitable non-natural interchain bonds include disulfide bonds, hydrogen bonds, electrostatic interactions, salt bridging or hydrophobic-hydrophilic interactions, mortar and pestle structures, or combinations thereof.

"disulfide" refers to a covalent bond having the structure R-S-S-R'. Cysteine this amino acid comprises a thiol group which may form a disulfide bond with a second thiol group, e.g. from another cysteine residue. Disulfide bonds may be formed between two cysteine residues located on two polypeptide chains, respectively, thereby forming an interchain bridge or interchain bond.

"unnatural" inter-chain bonds, as used herein, refer to inter-chain bonds that do not exist in the natural association of the native counterpart TCR constant region. For example, the non-native interchain bond may be formed between a mutated amino acid residue and a native amino acid residue each located on a separate TCR constant region, or between two mutated amino acid residues located on separate TCR constant regions. In certain embodiments, the at least one non-native inter-chain bond is formed between a first mutated residue comprised in a first TCR constant region of the polypeptide complex and a second mutated residue comprised in a second TCR constant region.

As used herein, the term "contact interface" refers to a specific region on a polypeptide where the polypeptides interact/associate with each other. The contact interface comprises one or more amino acid residues capable of interacting with corresponding amino acid residues that are in contact or associate when the interaction occurs. The amino acid residues in the contact interface may or may not be in a contiguous sequence. For example, when the interface is three-dimensional, amino acid residues within the interface may separate at different positions on the linear sequence.

Preparation method

The present disclosure provides isolated nucleic acids or polynucleotides encoding the polypeptide complexes and bispecific polypeptide complexes provided herein.

As used herein, the term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in single or double stranded form, and polymers thereof. Unless specifically limited, the term encompasses polynucleotides containing known analogs of natural nucleotides that have similar binding properties to a reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular polynucleotide sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is replaced with mixed bases and/or deoxyinosine residues (see Batzer et al, nucleic Acid Res.19:5081 (1991); ohtsuka et al, J.biol. Chem.260:2605-2608 (1985); and Rossolini et al, mol. Cell. Probes 8:91-98 (1994)).

Nucleic acids or polynucleotides encoding the polypeptide complexes and bispecific polypeptide complexes provided herein can be constructed using recombinant techniques. To this end, DNA encoding the antigen binding components (e.g., CDRs or variable regions) of the parent antibody can be isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of specifically binding to the genes encoding the heavy and light chains of the antibody). Likewise, DNA encoding the TCR constant region can be obtained. As an example, a polynucleotide sequence encoding a variable domain (VH) and a polynucleotide sequence encoding a first TCR constant region are obtained and operably linked to allow transcription and expression in a host cell to produce a first polypeptide. Similarly, a polynucleotide sequence encoding VL is operably linked to a polynucleotide sequence encoding a second TCR constant region so as to allow expression of a second polypeptide in the host cell. If desired, polynucleotide sequences encoding one or more spacers may also be operably linked to other coding sequences to allow for the expression of the desired product.

The coding polynucleotide sequence may optionally be further operably linked to one or more regulatory sequences in an expression vector such that expression or production of the first and second polypeptides is feasible and under suitable control.

The coding polynucleotide sequence may be inserted into a vector for further cloning (amplification of DNA) or expression using recombinant techniques known in the art. In another embodiment, the polypeptide complexes and bispecific polypeptide complexes provided herein can be produced by homologous recombination as known in the art. Many vectors are available. The components of the carrier include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter (e.g., SV40, CMV, EF-1. Alpha.) and a transcription termination sequence.

As used herein, the term "vector" refers to a cargo into which a polynucleotide encoding a protein may be operably inserted so as to cause expression of the protein. Typically, the construct further comprises suitable regulatory sequences. For example, the polynucleotide molecule may comprise regulatory sequences located in the 5' -flanking regions of the nucleotide sequence encoding the guide RNA and/or the nucleotide sequence encoding the site-directed modified polypeptide, which are operably linked to the coding sequence in a manner that enables expression of the desired transcript/gene in the host cell. The vector may be used to transform, transduce or transfect a host cell in order to cause expression of the genetic element it carries within the host cell. Examples of vectors include plasmids, phagemids, cosmids, artificial chromosomes such as Yeast Artificial Chromosomes (YACs), bacterial Artificial Chromosomes (BACs) or P1-derived artificial chromosomes (PACs), phages such as lambda phage or M13 phage, and animal viruses. Classes of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., SV 40). The vector may contain a variety of different elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selectable elements, and reporter genes. In addition, the vector may contain an origin of replication. The carrier may also include materials that facilitate its entry into the cell, including but not limited to viral particles, liposomes, or protein coatings.

In certain embodiments, the vector system includes mammalian, bacterial, yeast systems, and the like, and includes plasmids such as, but not limited to pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pCMV, pEGFP, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, psg L, pBABE, pWPXL, pBI, p TV-L, pPro18, pTD, pRS420, pLexA, pact2.2, and the like, as well as other laboratory and commercially available vectors. Suitable vectors may include plasmids or viral vectors (e.g., replication defective retroviruses, adenoviruses, and adeno-associated viruses).

Vectors comprising the polynucleotide sequences provided herein can be introduced into host cells for cloning or gene expression. As used herein, the phrase "host cell" refers to a cell into which an exogenous polynucleotide and/or vector has been introduced.

Suitable host cells for cloning or expressing the DNA in the vectors herein are prokaryotes, yeast or higher eukaryote cells as described above. Prokaryotes suitable for this purpose include eubacteria, e.g. gram-negative or gram-positive organisms, e.g. enterobacteriaceae, e.g. escherichia such as escherichia coli, enterobacteriaceae, erwinia, klebsiella, proteus, salmonella such as salmonella typhimurium, serratia such as serratia marcescens and shigella, and bacillus such as bacillus subtilis and bacillus licheniformis, pseudomonas such as pseudomonas aeruginosa and streptomyces.

In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are cloning or expression hosts suitable for vectors encoding polypeptide complexes and bispecific polypeptide complexes. Saccharomyces cerevisiae or Saccharomyces cerevisiae is the most commonly used lower eukaryotic host microorganism. However, a number of other genera, species and strains are commonly available and useful herein, such as schizosaccharomyces pombe, kluyveromyces hosts such as kluyveromyces lactis (k.lactis), kluyveromyces fragilis (k.fragilis) (ATCC 12,424), k.bulgaricus (ATCC 16,045), k.wickeramii (ATCC 24,178), k.walti (ATCC 56,500), k.drosophilarum (ATCC 36,906), k.thertolrans and k.marxians, yarrowia (EP 402,226), pichia pastoris (EP 183,070), candida, trichoderma reesei (EP 244,234), neurospora crassa, schwannoma hosts such as Schwanniomyces occidentalis, and fungi such as neurospora, penicillin, tortilla and aspergillus hosts such as aspergillus nidulans (a. Nidulans) and aspergillus niger (a. Nager).

Host cells suitable for expression of the glycosylated polypeptide complexes and bispecific polypeptide complexes provided herein are derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. A large number of baculovirus strains and variants and corresponding tolerant insect host cells from hosts such as spodoptera frugiperda (trichostrongyloides), aedes aegypti (mosquitoes), aedes albopictus (mosquitoes), drosophila melanogaster (drosophila) and bombyx mori have been identified. A variety of different viral strains for transfection are publicly available, for example the L-1 variant of the Spodoptera frugiperda NPV and the Bm-5 strain of the silkworm NPV, and such viruses may be used as herein viruses according to the invention, in particular for transfection of Spodoptera frugiperda cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be used as hosts.

However, the greatest interest is in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become routine. Examples of useful mammalian cell lines are monkey kidney CV1 cell lines transformed with SV40 (COS-7, ATCC CRL 1651), human embryonic kidney cell lines (293 or 293 cells subcloned for growth in suspension culture, graham et al, J.Gen Virol.36:59 (1977)) such as, for example, expi293, baby hamster kidney cells (BHK, ATCC CCL 10), chinese hamster ovary cells/-DHFR (CHO, urlaub et al, proc. Natl. Acad. Sci. USA 77:4216 (1980)), mouse sertoli cells (TM 4, mather, biol. Reprod.23:243-251 (1980)), monkey kidney cells (CV 1 ATCC CCL 70), african green monkey kidney cells (RO-76, ATCC CRL-1587), human cervical cancer cells (HELA, ATCC CCL 2), dog kidney cells (MDCK, ATCC CCL 34), bualo rat liver cells (BRL 3A, ATCC lung cancer cells (W4, SCL 138, CD 6, human liver tumor cells (Table 4, mather, mr. 3:39, B4, and Mather, mr. 3:38, F.3, B.6, and human tumor cells (1980)).

Host cells transformed with the above-described expression or cloning vectors may be cultured in conventional nutrient media suitably modified to induce promoters, select transformants, or amplify the cloning vectors.

To produce the polypeptide complexes and bispecific polypeptide complexes provided herein, host cells transformed with the expression vectors can be cultured in a variety of different media. Commercially available media such as Ham's F (Sigma), minimal Essential Media (MEM) (Sigma), RPMI-1640 (Sigma) and Dulbecco's Modified Eagle Medium (DMEM) (Sigma) are suitable for culturing host cells. Furthermore, any of the media described in Ham et al, meth.Enz.58:44 (1979), barnes et al, anal.biochem.102:255 (1980), U.S. Pat. Nos. 4,767,704, 4,657,866, 4,927,762, 4,560,655 or 5,122,469, WO 90/03430, WO 87/00195 or U.S. Pat.Re.30,985 may be used as the medium for the host cells. Any of these media may be supplemented, if desired, with hormones and/or other growth factors (e.g., insulin, transferrin or epidermal growth factor), salts (e.g., sodium chloride, calcium, magnesium, and phosphate), buffers (e.g., HEPES), nucleotides (e.g., adenosine and thymidine), antibiotics (e.g., gentamicin drugs), trace elements (defined as inorganic compounds typically present at a final concentration in the micromolar range), and glucose or equivalent energy sources. Any other desired supplement may also be included at suitable concentrations known to those skilled in the art. Culture conditions such as temperature, pH, etc., are conditions previously used for expression of the selected host cell and will be apparent to one of ordinary skill.

In certain embodiments, the polypeptide complex or bispecific polypeptide complex may be linked to the conjugate directly or indirectly, e.g., through another conjugate or through a linker. For example, a polypeptide complex or bispecific polypeptide complex with a reactive residue such as cysteine may be linked to a thiol-reactive reagent in which the reactive group is, for example, maleimide, iodoacetamide, pyridyl disulfide or other thiol-reactive coupling partner (Haugland, 2003, handbook of fluorescent Probes and research chemicals (Molecular Probes Handbook of Fluorescent Probes and Research Chemicals), molecular Probes, inc.; brinkley,1992,Bioconjugate Chem.3:2;Garman,1997, non-radioactive labeling: practical methods (Non-Radioactive Labelling: A Practical Approach), academic Press, london, means (1990) Bioconjugate chem.1:2; hermannson, G.; bioconjugate technology (Bioconjugate Techniques) (1996) Academic Press, san Diego, pp.40-55, 643-671).

As another example, the polypeptide complex or bispecific polypeptide complex may be conjugated to biotin and then indirectly conjugated to a second conjugate that is conjugated to avidin. As yet another example, the polypeptide complex or bispecific polypeptide complex may be linked to a linker, which is further linked to a conjugate. Examples of linkers include bifunctional coupling agents such as N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP) succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl diimidinate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azido compounds (e.g., bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (e.g., bis- (diazoniumbenzoyl) -ethylenediamine), diisocyanates (e.g., toluene 2, 6-diisocyanate), and histidine-active fluorine-containing compounds (e.g., 1, 5-difluoro-2, 4-dinitrobenzene). Particularly preferred coupling agents include N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP) (Carlsson et al biochem. J.173:723-737 (1978)) and N-succinimidyl-4- (2-pyridylthio) pentanoate (SPP) which provide disulfide bonds.

The conjugate may be a detectable label, a pharmacokinetic-modifying moiety, a purification moiety, or a cytotoxic moiety. Examples of detectable labels may include fluorescent labels (e.g., fluorescein, rhodamine, dansyl, phycoerythrin, or texas red), enzyme substrate labels (e.g., horseradish peroxidase, alkaline phosphatase, luciferase, glucoamylase, lysozyme, glycooxidase, or β -D-galactosidase), radioisotopes (e.g., 123I, 124I, 125I, 131I, 35S, 3H, 111In, 112In, 14C, 64Cu, 67Cu, 86Y, 88Y, 90Y, 177Lu, 211At, 186Re, 188Re, 153Sm, 212Bi, and 32P, other lanthanoids, luminescent labels), chromogenic moieties, digoxin, biotin/avidin, DNA molecules, or gold for detection. In certain embodiments, the conjugate may be a pharmacokinetic modifying moiety, such as PEG, that helps increase the half-life of the antibody. Other suitable polymers include, for example, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, ethylene glycol/propylene glycol copolymers, and the like. In certain embodiments, the conjugate may be a purification moiety such as a magnetic bead. A "cytotoxic moiety" may be any agent that is harmful to a cell or that can damage or kill a cell. Examples of cytotoxic moieties include, but are not limited to, paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthrax-dione, mitoxantrone, mithramycin, dactinomycin, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin and analogs thereof, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, dacarbazine), alkylating agents (e.g., mechlorethamine, thiotepa, chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C and cisplatin (II)), antimetabolites (e.g., mitomycin) and mitomycetin (e.g., mitomycin) and mitomycin (e.g., mitomycin) and procyanidins (AMC).

Methods for coupling conjugates to proteins such as antibodies, immunoglobulins, or fragments thereof can be found, for example, in U.S. Pat. No. 5,208,020, U.S. Pat. No. 6,441,163, WO2005037992, WO2005081711, and WO2006/034488, which are incorporated herein by reference in their entirety.

Pharmaceutical composition

The present disclosure also provides a pharmaceutical composition comprising a polypeptide complex or bispecific polypeptide complex provided herein and a pharmaceutically acceptable carrier.

The term "pharmaceutically acceptable" means that the carrier, medium, diluent, excipient and/or salt referred to is generally chemically and/or physically compatible with the other ingredients comprising the dosage form, and physiologically compatible with the recipient thereof.

By "pharmaceutically acceptable carrier" is meant an ingredient in a pharmaceutical dosage form other than the active ingredient that is biologically active and non-toxic to the subject. Pharmaceutically acceptable carriers for the pharmaceutical compositions disclosed herein can include, for example, pharmaceutically acceptable liquid, gel or solid carriers, aqueous media, non-aqueous media, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending/dispersing agents, sequestering or chelating agents, diluents, adjuvants, excipients or non-toxic auxiliary substances, other components known in the art, or various combinations thereof.

Therapeutic method

Also provided are methods of treatment, the methods comprising: a therapeutically effective amount of a polypeptide complex or bispecific polypeptide complex provided herein is administered to a subject in need thereof, thereby treating or preventing a condition or disorder. In certain embodiments, the subject has been identified as having a disorder or condition that is likely to respond to the polypeptide complexes or bispecific polypeptide complexes provided herein.

As used herein, the term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, e.g., non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. The terms "patient" or "subject" are used interchangeably unless otherwise indicated.

The terms "treatment" and "treatment method" refer to both therapeutic treatment and prophylactic measures. The individual in need of treatment may include individuals already with the particular medical disorder and those who are ultimately likely to suffer from the disorder.

In certain embodiments, the conditions and disorders include tumors and cancers, such as non-small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymus cancer, leukemia, lymphoma, myeloma, mycosis, merkel cell carcinoma and other hematological malignancies, such as Classical Hodgkin's Lymphoma (CHL), primary mediastinal large B-cell lymphoma, T-cell/tissue cell enriched B-cell lymphoma, EBV positive and negative PTLD, EBV related diffuse large B-cell lymphoma (DLBCL), plasmablastomal lymphoma, extralymph node NK/T-cell lymphoma, nasopharyngeal carcinoma, HHV8 related primary effusion lymphoma, hodgkin's lymphoma, central Nervous System (CNS) tumors such as primary CNS lymphoma, spinal cord axis tumors, brain stem glioma.

Examples

Example 1: preparation of research materials

1. Commercial materials

2. Production of antigens and other proteins

2.1 production of antigen

Nucleic acids encoding human PD-1, human and cynomolgus monkey LAG-3ECD (extracellular domain) were synthesized by Sangon Biotech. PD-1 or LAG-3 gene fragments were amplified from the synthesized nucleic acid and inserted into the expression vector pcDNA3.3 (ThermoFisher). The inserted PD-1 or LAG-3 gene fragments were further confirmed by DNA sequencing. Fusion proteins containing PD-1 or LAG-3 ECDs with various tags, including human Fc, mouse Fc, were obtained by transfection of the PD-1 or LAG-3 genes into 293F cells (ThermoFisher). The cells were expressed in FreeStyle 293 expression medium at 37℃in 5% CO ₂ And (5) culturing. After 5 days of culture, supernatants harvested from culture of transiently transfected cells were used for protein purification. The fusion proteins are purified by protein a and/or SEC columns. The unlabeled LAG-3ECD protein is produced by cleavage of an ECD-hFc fusion protein with a factor Xa protease. Purified proteins were used for screening and characterization.

Human PD-L1 ECD, human CTLA-4ECD and CD28 ECD were generated with mouse Fc tags as described above.

2.2 production of reference antibodies

The gene sequences of anti-human PD-1 or LAG-3 reference antibodies (LAG-3-BMK 1 and PD-1-BMK 1) were synthesized based on the information disclosed in patent applications US20110150892A1 (LAG-3-BMK 1 is referred to as "25F 7") and WO2006121168 (PD-1-BMK 1 is referred to as "5C 4"), respectively.

The sequences of anti-human PD-1 XLAG-3 reference antibodies BsAb-BMK1, bsAb-BMK2 and BsAb-BMK3 were synthesized based on the information disclosed in patent applications WO2015200119A8 (BsAb-BMK 1 referred to as "SEQ25& SEQ 27"), WO2017087589A2 (BsAb-BMK 2 referred to as "SEQ 110") and WO2015200119A8 (BsAb-BMK 3 referred to as "SEQ 5 and 4"), respectively. The synthesized gene sequence was incorporated into plasmid pcDNA3.3. Cells transfected with the plasmid were cultured for 5 days and the supernatant was collected for protein purification using a protein a column. The resulting reference antibodies were analyzed by SDS-PAGE and SEC and then stored at-80 ℃.

3. Construction of cell lines

ConstructionHuman, cynomolgus monkey PD-1 or LAG-3 transfected cell lines were used. Briefly, CHO-S or 293F cells were transfected with pcDNA3.3 expression vectors containing full-length human, cynomolgus PD-1 or LAG-3, respectively, using Lipofectamine transfection kit according to the manufacturer' S protocol. 48-72 hours after transfection, the transfected cells were cultured in medium containing blasticidin for selection and tested for target expression. Monoclonal cell lines expressing human PD-1 and monoclonal cell lines expressing cynomolgus monkey LAG-3 were obtained by limiting dilution.

Using Nucleofactor (Lonza), the Jurkat cell line was transfected with a plasmid containing the human full length PD-1/NFAT reporter. At 72 hours post-transfection, the transfected cells were cultured in medium containing hygromycin for selection and tested for expression of the target. After two months, jurkat cells expressing human PD-1 and stably integrated NFAT luciferase reporter gene were obtained.

Example 2: bispecific antibody production

1. Construction of expression vectors

DNA sequences encoding VH and VL of anti-PD-1 parent antibody were ligated to the N-terminus of anti-LAG-3 parent antibody. The CH1 and CL domains of the anti-PD-1 parent antibody Fab were replaced with TCR β and TCR α constant domains, respectively. The DNA sequences encoding the chimeric heavy chain fragments (anti-PD-1 VH and tcrp) were linked to the N-terminus of the heavy chain of the anti-LAG-3 parent antibody via a (G4S) x2 linker. Genes encoding anti-PD-1 chimeric light chains (VL and tcra), normal anti-LAG-3 antibody light chains, or the bispecific heavy chains described above, were cloned into modified pcdna3.3 expression vectors, respectively.

In addition, u6T1.G25-1.uIgG4.SP (YTE) was constructed, which introduced triple mutation M252Y/S254T/T256E (YTE) in the Fc portion of u6T1.G25-1.uIgG4.SP to increase the half-life of the antibody in serum.

2. Small scale transfection, expression and purification

Plasmids of bispecific antibodies were transfected into Expi293 cells. Cells were cultured for 5 days and the supernatant was collected for protein purification using a protein a column (GE Healthcare, 175438). The resulting antibodies were analyzed by SDS-PAGE and HPLC-SEC and then stored at-80 ℃.

3. Results

Sequence of lead candidate

The sequences of the antibody precursors are listed in table 2 and the CDRs are listed in table 1.

Example 4: in vitro characterization

Binding of PD-1 XLAG-3 bispecific antibodies to human PD-1 or LAG-3 proteins

Human PD-1 expressing cells or transiently transfected human LAG-3 expressing 293F cells were incubated with various concentrations of PD-1 XLAG-3 antibodies, respectively. Binding of the PD-1 XLAG-3 antibody to the cells was detected using PE-labeled goat anti-human IgG antibody. The MFI of the cells was measured by flow cytometry and analyzed by FlowJo (version 7.6.1).

As shown in FIG. 1 and Table 3, the primer PD-1 XLAG-3 was bispecificEC of antibodies binding to cell surface human PD-1 ₅₀ Corresponding to the reference antibody.

TABLE 3 EC of binding of PD-1 XLAG-3 bispecific antibodies to cell surface human PD-1 ₅₀

Antibodies to	EC 50 (nM)
		U6T1.G25-1.uIgG4.SP	1.13
PD-1-BMK1	0.33
		BsAb-BMK1	0.46
BsAb-BMK3	0.45

As shown in FIG. 2 and Table 4, the lead PD-1 XLAG-3 bispecific antibody binds to the EC of cell surface human LAG-3 ₅₀ Corresponding to the reference antibody.

TABLE 4 EC of PD-1 XLAG-3 bispecific antibody binding to cell surface human LAG-3 ₅₀

Antibodies to	EC 50 (nM)
		U6T1.G25-1.uIgG4.SP	3.76
LAG-3-BMK1	2.40
		BsAb-BMK3	0.96

Binding of PD-1 XLAG-3 bispecific antibodies to cynomolgus monkey PD-1 or LAG-3

For PD-1 binding, cynomolgus monkey PD-1 expressing 293F cells were incubated with various concentrations of PD-1 XLAG-3 antibodies, respectively. Binding of the PD-1 XLAG-3 antibody to the cells was detected using PE-labeled goat anti-human IgG antibody. The MFI of cells was measured by flow cytometry and analyzed by FlowJo.

For LAG-3 binding, the plates were coated with PD-1 XLAG-3 antibody overnight at 4 ℃. After blocking and washing, various concentrations of His-tagged cynomolgus LAG-3 protein were added to the plates and incubated for 1 hour at room temperature. Plates were then washed and subsequently incubated with HRP-labeled mouse anti-His antibody for 1 hour. After washing, TMB substrate was added and the color reaction was stopped by 2 MHCl. The absorbance at 450nm was read using a microplate reader.

As shown in fig. 3 and table 5, the leader PD-1×lag-3 bispecific antibody binds to EC of cell-surface cynomolgus monkey PD-1 ₅₀ Corresponding to the reference antibody.

TABLE 5 EC of PD-1 XLAG-3 bispecific antibody binding to cell-surface cynomolgus monkey PD-1 ₅₀

Antibodies to	EC 50 (nM)
		U6T1.G25-1.uIgG4.SP	0.50
PD-1-BMK1	0.28
		LAG-3-BMK3	0.33

As shown in FIG. 4 and Table 6, the lead PD-1 XLAG-3 bispecific antibody binds to cell-surface cynomolgus monkey LAG-3 EC ₅₀ Is superior to LAG-3-BMK1.

TABLE 6 EC of PD-1 XLAG-3 bispecific antibodies binding to cell-surface cynomolgus monkey LAG-3 protein ₅₀

Antibodies to	EC 50 (nM)
		U6T1.G25-1.uIgG4.SP	0.14
LAG-3-BMK1	Weak and weak
		BsAb-BMK3	0.33

Binding of PD-1 XLAG-3 bispecific antibodies to mouse PD-1 or LAG-3

For mouse PD-1 binding, the plates were coated with mouse Fc-tagged PD-1 overnight at 4 ℃. After blocking and washing, various concentrations of bispecific antibodies were added to the plates and incubated for 1 hour at room temperature. Plates were then washed and subsequently incubated with HRP-labeled goat anti-human IgG antibody for 1 hour. After washing, TMB substrate was added and the color reaction was stopped by 2M HCl. The absorbance at 450nm was read using a microplate reader.

For mouse LAG-3 binding, plates were coated with mouse anti-His antibody overnight at 4 ℃. After blocking and washing, his-tagged LAG-3 protein was added to the plates. The plates were incubated for 1 hour at room temperature after washing with various concentrations of PD-1 XLAG-3 antibodies. Plates were then washed and subsequently incubated with HRP-labeled goat anti-human IgG antibody for 1 hour. After washing, TMB substrate was added and the color reaction was stopped by 2M HCl. The absorbance at 450nm was read using a microplate reader.

As shown in fig. 5A and 5B, the lead PD-1×lag-3 bispecific antibody did not bind to mouse PD-1 or LAG-3.

4. Cross-reactivity with human CD4, CTLA-4 and CD28

Cross-reactivity with human CD4, CTLA-4 or CD28 was measured by ELISA. Plates were coated with 1. Mu.g/mL human CD4, CTLA-4 or CD28 overnight at 4 ℃. After blocking and washing, various concentrations of PD-1XLAG-3 antibodies were added to the plate and incubated for 1h at room temperature. The plates were then washed and then incubated with the corresponding secondary antibodies for 60min. After washing, TMB substrate was added and the color reaction was stopped by 2M HCl.

The results in FIGS. 6A, 6B and 6C indicate that PD-1XLAG-3 bispecific antibodies do not bind to human CTLA-4, CD28 or CD4 proteins.

5. Affinity for human, mouse, cynomolgus monkey PD-1 and LAG-3 by SPRForce of forceTest

The binding affinity of the bispecific antibody to the antigen was determined by SPR assay using Biacore 8K. The PD-1xLAG-3 antibody was captured on a CM5 sensor chip (GE) immobilized with an anti-human IgG Fc antibody. His-tagged human PD-1 protein (MW: 40 KD) and cynomolgus PD-1 (MW: 40 KD) were injected onto the sensor chip at different concentrations at a flow rate of 30. Mu.L/min for a binding period of 120s followed by dissociation of 800 s.

For affinity to human LAG-3, PD-1xLAG-3 antibodies were immobilized on CM5 sensor chips. Using the single cycle kinetic method, unlabeled human LAG-3 was injected onto the sensor chip at different concentrations at a flow rate of 30 μL/min for a binding period of 180s followed by 3600s dissociation. The chip was regenerated with 10mM glycine (pH 1.5).

The sensorgram for the blank surface and buffer channel was subtracted from the test sensorgram. Experimental data were fitted in a 1:1 model using langmuir analysis.

TABLE 7 affinity of PD-1XLAG-3 bispecific antibodies for human PD-1

Ab	ka(1/Ms)	kd(1/s)	KD(M)
				U6T1.G25-1.uIgG4.SP	1.20E+06	1.19E-03	9.88E-10
PD-1-BMK1	4.02E+05	1.35E-03	3.37E-09
				BsAb-BMK3	3.80E+05	1.36E-03	3.58E-09

TABLE 8 affinity of PD-1XLAG-3 bispecific antibodies for human LAG-3

Ab	ka(1/Ms)	kd(1/s)	KD(M)
				U6T1.G25-1.uIgG4.SP	5.90E+05	<1.00E-05	<1.70E-11
PD-1-BMK1	4.87E+05	3.34E-04	6.85E-10
				BsAb-BMK3	1.02E+07	8.70E-04	8.51E-11

Dual binding of PD-1XLAG-3 bispecific antibodies to human PD-1 and LAG-3 proteins

Plates were coated with 1. Mu.g/mL human PD-1 tagged with mouse Fc at 4℃overnight. After blocking and washing, various concentrations of PD-1×lag-3 antibodies were added to the plates and incubated for 1 hour at room temperature. The plates were then washed and then incubated with His-tagged LAG-3 protein for 1 hour. After washing, HRP-labeled anti-His antibody was added to the plate and incubated for 1 hour at room temperature. After washing the TMB substrate was added and the color reaction was stopped by 2M HCl. The absorbance at 450nm was read using a microplate reader.

As shown in FIG. 7 and Table 9, the EC of binding of the primer PD-1 XLAG-3 bispecific antibody to LAG-3 protein ₅₀ Is equivalent to and better than BsAb-BMK1 and BsAb-BMK2.

TABLE 9 EC of PD-1 XLAG-3 bispecific antibodies binding to human PD-1 and LAG-3 proteins ₅₀

Blocking of binding of PD-L1 protein to PD-1 expressing cells

Antibodies were serially diluted in 1% BSA-PBS and mixed with the mouse Fc tagged PD-L1 protein at 4 ℃. The mixture was transferred to 96-well plates seeded with CHO-S cells expressing PD-1. Binding of PD-L1 protein to cells expressing PD-1 was detected using goat anti-mouse IgG Fc-PE antibodies. MFI was assessed by flow cytometry and analyzed by FlowJo software.

As shown in fig. 8 and table 10, the primer PD-1×lag-3 bispecific antibody blocks EC of binding of PD-1 to PD-L1 expressing cells ₅₀ Corresponding to the reference antibody.

TABLE 10 EC of PD-1 XLAG-3 bispecific antibody blocking the binding of PD-1 to PD-L1 ₅₀

Antibodies to	EC 50 (nM)
		U6T1.G25-1.uIgG4.SP	1.34
PD-1-BMK1	0.22
		BsAb-BMK3	0.33

Blocking of binding of LAG-3 protein to MHC-II expressed on Raji cells

Antibodies were serially diluted in complete medium and mixed with LAG-3 protein with mouse Fc tag at 4 ℃. The mixture was transferred to 96-well plates seeded with Raji cells expressing MHC-II on the surface. Binding of LAG-3 protein to Raji cells was detected using goat anti-mouse IgG Fc-PE antibody. MFI was assessed by flow cytometry and analyzed by FlowJo software.

As shown in FIG. 9 and Table 11, the primer PD-1 XLAG-3 bispecific antibody blocked EC of binding of LAG-3 to MHC-II expressing Raji cells ₅₀ Better than BsAb-BMK1 and BsAb-BMK2 and comparable to other reference antibodies.

TABLE 11 EC of PD-1 XLAG-3 bispecific antibody blocking binding of LAG-3 to MHC-II ₅₀

PD-1 XLAG-3 bispecific antibody against Jurkat expressing PD-1 and LAG-3 with NFAT reporter Influence of (2)

The whole human LAG-3 plasmid was transiently transfected into Jurkat cells expressing human PD-1 and stably integrated NFAT luciferase reporter. After 48 hours, the cells were seeded with Raji cells in 96-well plates in the presence of SEE (staphylococcal enterotoxin E). Test antibodies are added to the cells. The plates were incubated at 37℃with 5% CO ₂ Incubate overnight. After incubation, the reconstituted luciferase substrate One-Glo was added and luciferase intensity was measured by a microplate spectrophotometer.

As demonstrated in FIG. 10, antibodies enhanced the NFAT pathway of Jurkat expressing PD-1 and LAG-3 in a reporter gene assay. Fold higher than the combination of PD-1-BMK1 and LAG-3-BMK1, as well as other reference antibodies.

Effect of PD-1 XLAG-3 bispecific antibodies on human allogeneic Mixed Lymphocyte Reaction (MLR)

Human Peripheral Blood Mononuclear Cells (PBMCs) were freshly isolated from healthy donors using Ficoll-Paque PLUS gradient centrifugation. The human monocyte enrichment kit was used to isolate monocytes according to the manufacturer's instructions. Cells were cultured in medium containing GM-CSF and IL-4 for 5 to 7 days to produce Dendritic Cells (DCs). Use of human CD4 ⁺ T cell enrichment kit for isolation of human CD4 according to manufacturer's protocol ⁺ T cells. Purified CD4 ⁺ T cells were co-cultured with allogeneic Immature DCs (iDC) in 96-well plates in the presence of various concentrations of PD-1×lag-3 antibodies. The plates were incubated at 37℃with 5% CO ₂ Incubation was performed. Supernatants were harvested on day 3 and day 5, respectively, for IL-2 and IFN-gamma testing.Human IL-2 and IFN-gamma release was measured by ELISA using matched antibody pairs. Recombinant human IL-2 and IFN-gamma were used as standards, respectively. Plates were pre-coated with capture antibodies specific for human IL-2 or IFN-gamma, respectively. After blocking, 50 μl of standard or sample was pipetted into each well and incubated for 2 hours at ambient temperature. After removal of unbound material, biotin-conjugated detection antibodies specific for the corresponding cytokines were added to the wells and incubated for 1 hour. HRP-labeled streptavidin was then added to the wells and incubated for 30 minutes at ambient temperature. The color development was performed by adding 50. Mu.L of TMB substrate, followed by termination of the reaction with 50. Mu.L of 2N HCl. Absorbance at 450nm was read using a microplate spectrophotometer.

As demonstrated in fig. 11A and 11B, the lead antibodies enhanced IL-2 and IFN- γ secretion in mixed lymphocyte reactions.

11. Thermal stability test by Differential Scanning Fluorometry (DSF)

Tm of antibodies was investigated using the quantsudio 7Flex real-time PCR system (Applied Biosystems). mu.L of antibody solution was mixed with 1. Mu.L of 62.5 XSYPRO Orange solution (Invitrogen) and transferred to a 96-well plate. The plate was heated from 26 ℃ to 95 ℃ at a rate of 0.9 ℃/min and the resulting fluorescence data collected. The negative derivative of the fluorescence change at different temperatures is calculated and the maximum is defined as the melting temperature Tm. If the protein has multiple unfolding transitions, the first two Tms, designated Tm1 and Tm2, respectively, are reported. The data acquisition and Tm calculation are automated by the operating software.

TABLE 12 Tm of PD-1 XLAG-3 bispecific antibodies

Example 5: in vivo characterization

In vivo anti-tumor Activity of PD-1 XLAG-3 antibodies

Evaluation of PD-1 XL using human PD-1/LAG-3 transgenic (knock-in) mice (Biocytogen) and B16F10 tumor modelsAG-3 antibodies inhibit tumor cell growth in vivo. On day 0, mice were treated with 1X 10 ⁶ The melanoma cells B16F10 of individual mice are implanted subcutaneously and reach 50-60mm in the tumor ³ Mice were grouped (n=8).

On days 0, 3, 6, 9, 12 and 15, mice were intraperitoneally treated with PD-1mAb alone (PD-1-BMK 1) (10 mg/kg), LAG-3mAb alone (LAG-3-BMK 1) (10 mg/kg), PD-1 XLAG-3 antibody U6T1.G25-1.UIgG4.SP (13.1 mg/kg) or a combination of PD-1-BMK1 (10 mg/kg) and LAG-3-BMK1 (10 mg/kg). Human IgG4 isotype control antibody (10 mg/kg) was provided as a negative control.

Tumor volumes and animal body weights were measured within 2 weeks after injection. Tumor volume in mm using the following formula ³ The unit is expressed as: v=0.5ab ² Wherein a and b are the long and short diameters of the tumor, respectively.

Tumor volume and survival curves of treated mice are shown in fig. 12A and 12B. The results showed that in hLAG-3/hPD-1 knock-in mice, treatment with LAG-3BMK1 or PD-1BMK1 antibodies had little effect on B16F10 tumor growth inhibition, while u6t1.g25-1.uigg4.sp caused stronger tumor growth inhibition compared to LAG-3BMK1 alone or PD-1BMK1 alone. The potency of u6t1.G25-1.Uigg4.Sp was comparable to the combination of PD-1 and LAG-3 antibodies. Meanwhile, in fig. 12B, weight gain of each group indicates good safety without significant toxicity.

Sequence listing

<110> Shanghai pharmaceutical Biotechnology Co., ltd

<120> bispecific antibodies against PD-1 and LAG-3

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Ser Tyr Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu

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Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

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Ser Leu Lys Leu Asn Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

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Ser Thr Ile Thr Gly Gly Gly Gly Ser Ile Tyr Tyr Ala Asp Ser Val

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Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

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Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

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Ala Lys Asn Arg Ala Gly Glu Gly Tyr Phe Asp Tyr Trp Gly Gln Gly

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Thr Leu Val Thr Val Leu

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Asp Tyr Val Ala Trp Tyr Gln Gln His Pro Gly Lys Val Pro Lys Leu

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Met Ile Tyr Asp Val Ser Glu Arg Pro Ser Gly Val Ser Asn Arg Phe

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Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu

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Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Thr

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Thr Thr Leu Val Val Phe Gly Gly Gly Thr Lys Leu Ser Val Leu

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Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Ala Gln Ala Gly

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Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

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Ser Thr Ile Thr Gly Gly Gly Gly Ser Ile Tyr Tyr Ala Asp Ser Val

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Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

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Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

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Ala Lys Asn Arg Ala Gly Glu Gly Tyr Phe Asp Tyr Trp Gly Gln Gly

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Thr Leu Val Thr Val Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu

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Val Ala Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys

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Ala Thr Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu

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Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Cys Thr

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Asp Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Gln Asp Ser Arg Tyr

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Ala Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro

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Arg Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn

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Asp Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser

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Ala Glu Ala Trp Gly Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

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Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

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Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Asp Ser Ile Ser Ser Thr

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Ser Tyr Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu

290 295 300

Trp Ile Gly Ser Phe Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

305 310 315 320

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

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Ser Leu Lys Leu Asn Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

340 345 350

Cys Ala Arg Met Gln Leu Trp Ser Tyr Asp Val Asp Val Trp Gly Gln

355 360 365

Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

370 375 380

Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala

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Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

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Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

420 425 430

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

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Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys

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Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro

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Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val

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Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

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Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

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Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser

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Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

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Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

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Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

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Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

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Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

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Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

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Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

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His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly

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Ser Tyr Glu Leu Thr Gln Pro Leu Ser Val Ser Val Ala Leu Gly Gln

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His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

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Arg Asp Ser Asn Arg Pro Ser Gly Ile Pro Glu Gly Phe Ser Gly Ser

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Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Ala Gln Ala Gly

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Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ile Trp Val Phe

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Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val

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Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr Gln Val Ser Gln Ser Lys

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Asp Ser Asp Val Tyr Ile Thr Asp Lys Cys Val Leu Asp Met Arg Ser

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Met Asp Phe Lys Ser Asn Ser Ala Val Ala Trp Ser Gln Lys Ser Asp

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Phe Phe Pro Ser Pro Glu Ser Ser

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Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln

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Asp Tyr Val Ala Trp Tyr Gln Gln His Pro Gly Lys Val Pro Lys Leu

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Met Ile Tyr Asp Val Ser Glu Arg Pro Ser Gly Val Ser Asn Arg Phe

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Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu

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Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Thr

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Thr Thr Leu Val Val Phe Gly Gly Gly Thr Lys Leu Ser Val Leu Gly

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Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu

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Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe

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Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val

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Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys

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Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser

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His Lys Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu

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Lys Thr Val Ala Pro Thr Glu Cys Ser

210 215

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<212> PRT

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<223> first polypeptide of WBP3658-U6T1.G25-1.UIgG4.SP (YTE)

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Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

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Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser His

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Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

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Ser Thr Ile Thr Gly Gly Gly Gly Ser Ile Tyr Tyr Ala Asp Ser Val

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Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

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Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

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Ala Lys Asn Arg Ala Gly Glu Gly Tyr Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu

115 120 125

Val Ala Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys

130 135 140

Ala Thr Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu

145 150 155 160

Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Cys Thr

165 170 175

Asp Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Gln Asp Ser Arg Tyr

180 185 190

Ala Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro

195 200 205

Arg Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn

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Asp Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser

225 230 235 240

Ala Glu Ala Trp Gly Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

245 250 255

Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

260 265 270

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Asp Ser Ile Ser Ser Thr

275 280 285

Ser Tyr Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu

290 295 300

Trp Ile Gly Ser Phe Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

305 310 315 320

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

325 330 335

Ser Leu Lys Leu Asn Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

340 345 350

Cys Ala Arg Met Gln Leu Trp Ser Tyr Asp Val Asp Val Trp Gly Gln

355 360 365

Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

370 375 380

Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala

385 390 395 400

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

405 410 415

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

420 425 430

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

435 440 445

Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys

450 455 460

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro

465 470 475 480

Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val

485 490 495

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile Thr Arg Glu

500 505 510

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

515 520 525

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

530 535 540

Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser

545 550 555 560

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

565 570 575

Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

580 585 590

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

595 600 605

Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

610 615 620

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

625 630 635 640

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

645 650 655

Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg

660 665 670

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

675 680 685

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

690 695 700

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Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val Phe Glu Pro Ser

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Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys Leu Ala

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Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp Trp Val Asn Gly

35 40 45

Lys Glu Val His Ser Gly Val Cys Thr Asp Pro Gln Pro Leu Lys Glu

50 55 60

Gln Pro Ala Leu Gln Asp Ser Arg Tyr Ala Leu Ser Ser Arg Leu Arg

65 70 75 80

Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe Arg Cys Gln

85 90 95

Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln Asp Arg

100 105 110

Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp Gly Arg

115 120 125

<210> 22

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<213> Artificial work

<220>

<223> engineering of the TCR alpha Domain

<400> 22

Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys Ser

1 5 10 15

Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr Gln

20 25 30

Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Cys Val

35 40 45

Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala Trp

50 55 60

Ser Gln Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Gln Asn Ser Ile

65 70 75 80

Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser

85 90

<210> 23

<211> 10

<212> PRT

<213> Artificial work

<220>

<223> linker between TCR β domain and VH of anti-LAG-3 binding domain

<400> 23

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210> 24

<211> 1

<212> PRT

<213> Artificial work

<220>

<223> linker between TCR β domain and VH of anti-PD-1 binding domain

<400> 24

Glu

1

<210> 25

<211> 2

<212> PRT

<213> Artificial work

<220>

<223> linker between VL and TCR alpha domain of anti-PD-1 binding domain

<400> 25

Pro Asp

1

<210> 26

<211> 93

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 26

Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys Ser

1 5 10 15

Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr Asn

20 25 30

Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Thr Val

35 40 45

Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala Trp

50 55 60

Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser Ile

65 70 75 80

Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser

85 90

<210> 27

<211> 127

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 27

Asp Leu Lys Asn Val Phe Pro Pro Lys Val Ala Val Phe Glu Pro Ser

1 5 10 15

Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys Leu Ala

20 25 30

Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp Trp Val Asn Gly

35 40 45

Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Pro Leu Lys Glu

50 55 60

Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser Ser Arg Leu Arg

65 70 75 80

Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe Arg Cys Gln

85 90 95

Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln Asp Arg

100 105 110

Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp Gly Arg

115 120 125

Claims (29)

1. A bispecific antibody or antigen-binding fragment thereof comprising:

a first polypeptide chain comprising, from N-terminus to C-terminus, a first heavy chain Variable (VH) domain of a PD-1 antibody, a first T Cell Receptor (TCR) constant region operably linked to a second VH domain of a LAG-3 antibody and antibody heavy chain constant regions CH1, CH2 and CH3 domain,

a second polypeptide chain comprising, from N-terminus to C-terminus, a first light chain Variable (VL) domain of a PD-1 antibody and an operably linked second TCR constant region,

a third polypeptide chain comprising, from N-terminus to C-terminus, a second VL domain of a LAG-3 antibody and an operably linked antibody light chain Constant (CL) domain,

wherein the first TCR constant region and the second TCR constant region are capable of forming dimers comprising at least one unnatural inter-chain disulfide bond,

Wherein the second VH domain of the LAG-3 antibody comprises H-CDR1, H-CDR2, and H-CDR3; wherein the H-CDR3 is as set forth in SEQ ID NO:1 is shown in the specification; the H-CDR2 is shown in SEQ ID NO:2 is shown in the figure; the H-CDR1 is shown in SEQ ID NO: as shown in figure 3, the number of the holes in the steel plate is,

wherein the second VL domain of the LAG-3 antibody comprises L-CDR1, L-CDR2, and L-CDR3; wherein the L-CDR3 is as shown in SEQ ID NO: shown in figure 7; the L-CDR2 is shown in SEQ ID NO: shown as 8; the L-CDR1 is shown in SEQ ID NO: as shown in the drawing 9,

wherein the first VH domain of the PD-1 antibody comprises H-CDR1, H-CDR2, H-CDR3; wherein the H-CDR3 is as set forth in SEQ ID NO:4 is shown in the figure; the H-CDR2 is shown in SEQ ID NO:5 is shown in the figure; the H-CDR1 is shown in SEQ ID NO: as shown in figure 6, the number of the holes in the steel plate,

wherein the first VL domain of the PD-1 antibody comprises L-CDR1, L-CDR2, and L-CDR3; wherein the L-CDR3 is as shown in SEQ ID NO:10 is shown in the figure; the L-CDR2 is shown in SEQ ID NO: 11; the L-CDR1 is shown in SEQ ID NO: shown at 12.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof provides a bivalent binding site specific for PD-1 or LAG-3.

3. The antibody or antigen-binding fragment thereof of claim 2, wherein the antibody or antigen-binding fragment thereof comprises two of the first polypeptide chains, two of the second polypeptide chains, and two of the third polypeptide chains.

4. The antibody or antigen-binding fragment thereof of claim 1, wherein the second VH domain of the LAG-3 antibody is set forth in SEQ ID NO: shown at 13.

5. The antibody or antigen-binding fragment thereof of claim 1, wherein the first VH domain of the PD-1 antibody is set forth in SEQ ID NO: 14.

6. The antibody or antigen-binding fragment thereof of claim 1, wherein the second VL domain of the LAG-3 antibody is set forth in SEQ ID NO: 15.

7. The antibody or antigen-binding fragment thereof of claim 1, wherein the first VL domain of the PD-1 antibody is set forth in SEQ ID NO: shown at 16.

8. The antibody or antigen binding fragment thereof of claim 1, wherein the first TCR constant region comprises an engineered TCR β constant region, relative to the sequence set forth in SEQ ID NO:27, which engineered TCR β constant region introduced mutated residues of K9E, S56C, N69Q and C74A.

9. The antibody or antigen-binding fragment thereof of claim 1, wherein the second TCR constant region comprises an engineered TCR alpha constant region, relative to the sequence set forth in SEQ ID NO:26, which engineered TCR a constant region introduced mutated residues of N32Q, T47C, N Q and N77Q.

10. The antibody or antigen binding fragment thereof of claim 8, wherein the engineered TCR β constant region is as set forth in SEQ ID NO: 21.

11. The antibody or antigen binding fragment thereof of claim 9, wherein the engineered TCR a constant region is as set forth in SEQ ID NO: shown at 22.

12. The antibody or antigen-binding fragment thereof of claim 1, wherein the first TCR constant region and the second VH domain are encoded by SEQ ID NO:23, and a peptide sequence of 23.

13. The antibody or antigen-binding fragment thereof of claim 1, wherein the first polypeptide further comprises an IgGFc fragment, wherein the IgGFc fragment is operably linked to a CH1 domain.

14. The antibody or antigen-binding fragment thereof of claim 1, wherein the first polypeptide is as set forth in SEQ ID NO:17 or 20.

15. The antibody or antigen-binding fragment thereof of claim 1, wherein the second polypeptide chain is as set forth in SEQ ID NO: shown at 18.

16. The antibody or antigen-binding fragment thereof of claim 1, wherein the third polypeptide chain is as set forth in SEQ ID NO: 19.

17. The antibody or antigen-binding fragment thereof of any one of claims 1-16, wherein the antibody or antigen-binding fragment

a) At 9.88×10 ^-10 Or lower K _D Binds to human PD-1; and is also provided with

b) At 1.70X10 ^-11 Or lower K _D Binds to human LAG-3.

18. The antibody or antigen-binding fragment thereof of any one of claims 1-16, wherein the antibody is a humanized antibody.

19. A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of any one of claims 1-16.

20. A cloning or expression vector comprising the nucleic acid molecule of claim 19.

21. A host cell comprising one or more cloning or expression vectors according to claim 20.

22. A method of producing the antibody or antigen-binding fragment thereof of any one of claims 1-16, the method comprising culturing the host cell of claim 21 and isolating the antibody.

23. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-16 and one or more pharmaceutically acceptable carriers.

24. An immunoconjugate comprising the antibody or antigen-binding fragment thereof of any one of claims 1-16 linked to a therapeutic agent.

25. A pharmaceutical composition comprising the immunoconjugate of claim 24 and one or more pharmaceutically acceptable carriers.

26. The pharmaceutical composition of claim 23 or 25, wherein the carrier comprises an excipient and a diluent.

27. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-16 for the manufacture of a medicament for the treatment or prevention of an immune disorder or cancer.

28. The use of claim 27, wherein the cancer is a cell of a cancer selected from the group consisting of melanoma, renal cancer, prostate cancer, breast cancer, colon cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, uterine cancer, ovarian cancer, and rectal cancer.

29. The use of claim 27, wherein the cancer is malignant melanoma of the skin or the eye.