5T4 / CD40 bispecific antibody

Abstract

The present invention provides CD40 agonist antibodies that have substantially higher CD40 activation activity in the presence of simultaneous tumor-associated antigen (TAA) binding than in the absence of such simultaneous binding. Such TAA-dependent CD40 agonism results in a greatly improved therapeutic index, reducing or completely eliminating adverse events typically associated with CD40 activation, such as cytokine release syndrome or hepatotoxicity. Also provided are, e.g., 5T4 / CD40 bispecific and multispecific antibodies and polypeptides, such as chimeric antigen receptors, which are optionally incorporated into these antibodies along with an anti-TAA unit. Also provided are methods of using these antibodies or polypeptides to treat and diagnose diseases, such as cancer.

Description

[0001] This application is a divisional application of the application patent application with application number 202380030002.2, entered into the Chinese national phase on September 24, 2024, with an application date of February 21, 2023, and with the title “Tumor Antigen Dependent CD40 Agonist Antibodies”. BACKGROUND

[0002] CD40 (tumor necrosis factor receptor superfamily 5, or cluster of differentiation 40) is a member of the TNF receptor superfamily (TNFRSF) and is a costimulatory molecule expressed on antigen-presenting cells (APCs) such as dendritic cells (DCs), B cells, and macrophages, as well as on non-immune cells and tumors. CD40 plays an important role in modulating the activity of APCs and in linking innate and adaptive immunity. The CD40 pathway is not only required for efficient T cell and B cell immune responses, but also provides a critical initial step in the development of both humoral and cellular immunity.

[0003] Upon activation, CD40 can induce dendritic cells to promote anti-tumor T cell activation and re-induce macrophages to destroy tumor stroma. Activation of CD40 has been used in combination with other therapies for treating cancer, such as immune checkpoint inhibitors. The combination of CD40 activation after chemotherapy acts as an in situ vaccine. In addition, CD40-activated macrophages are reported to rapidly infiltrate tumors and promote depletion of tumor stroma, and further enhance chemotherapy delivery. In summary, CD40 activation is an important mechanism that helps to transform so-called cold tumors into hot tumors.

[0004] Multiple approaches have been developed to activate CD40 in cancer patients. The initial CD40 therapeutic agonists were based on multimeric forms of its ligand, CD40L. Subsequently, the approach was mainly based on agonist CD40 antibodies, which were designed to mimic CD40L by cross-linking CD40. One of the most widely studied antibodies is selicrelumab (Roche), formerly known as CP-870,893 and RO7009789, which is a fully human IgG2 mAb. Other antibodies include CDX-1140 (Celldex), APX005M (Apexigen), SEA-CD40 (Seattle Genetics), ChiLob7 / 4 (University of Southampton), and ADC-1013 (Janssen / Alligator). CD40 antibodies vary in activation potency, ranging from very high (APX005M), high (selicrelumab), to weak (SEA-CD40). Some CD40 mAbs block the CD40L binding site, such as APX005M, while others (e.g., selicrelumab and CDX-1140) do not.

[0005] Clinical studies of these CD40 agonists have revealed a common set of dose-dependent adverse events. Among them, the main adverse event is cytokine release syndrome (CRS), which is characterized by various combinations of chills, stiffness, rash, nausea, fever, vomiting, muscle pain and back pain. Another major safety issue is dose-related hematological toxicity, such as decreases in peripheral lymphocytes, monocytes and platelets. Such reported toxicities are considered to be a legacy of CD40 agonist therapy and have prevented experts from advancing the clinical development of these candidates. There is an urgent need to develop CD40 agonist antibodies with effective therapeutic effects without the associated toxicities. Summary of the Invention

[0006] As presented, existing CD40 agonist antibodies, despite having acceptable CD40 activation efficacy, are associated with common adverse events such as cytokine release syndrome (CRS). These adverse effects are intrinsically linked to the biological mechanisms of CD40 agonism, and therefore controlling them without sacrificing therapeutic efficacy is challenging.

[0007] However, through careful design and selection, the inventors have identified new CD40 agonist Nanobodies that have significantly reduced CD40 activation capacity compared to existing antibodies (such as selumetumab). However, when used in a bispecific or multispecific format, the newly identified antibodies exhibit potent activation activity, which also includes an antibody portion that targets a tumor-associated antigen (TAA) expressed on target cells. Therefore, the TAA dependence of the newly identified antibodies allows these new antibodies to exhibit high therapeutic activity where activity is needed (e.g., at the target tumor site), while exhibiting lower activity or even no activity elsewhere. This latter property can therefore reduce or even eliminate the adverse effects that are typically associated with other CD40 agonist antibodies.

[0008] Therefore, according to one embodiment of the present disclosure, a single domain antibody or a polypeptide comprising the single domain antibody is provided, wherein the single domain antibody has binding specificity to human cluster of differentiation 40 (CD40) protein and comprises complementarity determining region 1 (CDR1), CDR2 and CDR3.

[0009] In some embodiments, the CDR1, CDR2, and CDR3 comprise (1) the amino acid sequences of SEQ ID NOs: 14, 15, and 16, respectively; (1a) the amino acid sequences of SEQ ID NOs: 14, 63, and 16; (1b) the amino acid sequences of SEQ ID NOs: 14, 64, and 16; (2) the amino acid sequences of SEQ ID NOs: 17, 18, and 19; (3) the amino acid sequences of SEQ ID NOs: 20, 21, and 22; (4) the amino acid sequences of SEQ ID NOs: 23, 24, and 25; (5) the amino acid sequences of SEQ ID NOs: 26, 27, and 28; (6) the amino acid sequences of SEQ ID NOs: 29, 30, and 31; (7) the amino acid sequences of SEQ ID NOs: 32, 33, and 34; (8) the amino acid sequences of SEQ ID NOs: 35, 36, and 37; (9) the amino acid sequences of SEQ ID NOs: 38, 39, and 40; (10) the amino acid sequences of SEQ ID NOs: 39, 41, and 42. (11) the amino acid sequence of SEQ ID NO:44, 45 and 46; (12) the amino acid sequence of SEQ ID NO:47, 48 and 49; or (13) the amino acid sequence of SEQ ID NO:50, 51 and 52.

[0010] In some embodiments, the CDR1 comprises the amino acid sequence of SEQ ID NO: 14, the CDR2 comprises the amino acid sequence of SEQ ID NO: 15, 63, or 64, and the CDR3 comprises the amino acid sequence of SEQ ID NO: 16.

[0011] In some embodiments, the CDR1 comprises the amino acid sequence of SEQ ID NO: 14, the CDR2 comprises the amino acid sequence of SEQ ID NO: 15, and the CDR3 comprises the amino acid sequence of SEQ ID NO: 16. In some embodiments, the antibody or polypeptide comprises an amino acid sequence selected from SEQ ID NO: 53, 54, 57, and 60.

[0012] In some embodiments, the CDR1 comprises the amino acid sequence of SEQ ID NO: 14, the CDR2 comprises the amino acid sequence of SEQ ID NO: 63, and the CDR3 comprises the amino acid sequence of SEQ ID NO: 16. In some embodiments, the antibody or polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 55, 58, and 61.

[0013] In some embodiments, the CDR1 comprises the amino acid sequence of SEQ ID NO: 14, the CDR2 comprises the amino acid sequence of SEQ ID NO: 64, and the CDR3 comprises the amino acid sequence of SEQ ID NO: 16. In some embodiments, the antibody or polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 56, 59, and 62.

[0014] In one embodiment, multispecific antibodies are also provided, comprising an antibody of the present disclosure and a second antibody or antigen-binding fragment having binding specificity for a second target antigen other than CD40. In some embodiments, the second target antigen is a tumor-associated antigen (TAA).

[0015] Another embodiment provides a multispecific antibody comprising a first antibody or antigen-binding fragment having binding specificity for human CD40 and a second antibody or antigen-binding fragment having binding specificity for a second target antigen, which is a tumor-associated antigen (TAA), wherein the multispecific antibody more effectively activates CD40 on target cells expressing the TAA as compared to CD40 on reference cells that do not express the TAA.

[0016] Another embodiment provides a multispecific antibody comprising a first antibody or antigen-binding fragment having binding specificity for human CD40 and a second antibody or antigen-binding fragment having binding specificity for a second target antigen that is a tumor-associated antigen (TAA), wherein the multispecific antibody does not activate CD40 on reference cells that do not express TAA.

[0017] In some embodiments, the multispecific antibody activates CD40 on target cells expressing the TAA at least 2-fold, or 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold more effectively than CD40 on reference cells that do not express the TAA. In some embodiments, the activation is measured using a multispecific antibody concentration of 0.001 nM to 200 nM, preferably 0.1 nM to 100 nM. In some embodiments, the activation is measured using a panel of CD40 functional assays, such as an NFκB reporter gene assay, an IL-12 secretion assay, a CD80 expression assay, a CD86 expression assay, or a Ki67 expression assay, or a Ki67 / CD86 expression assay.

[0018] The present disclosure provides conditionally activated CD40 bispecific antibodies that activate only in the presence of tumor cells expressing 5T4. 5T4 is an oncofetal protein that is rarely expressed in normal adult tissues, however, its expression is upregulated in various cancers. CD40 cross-linking via 5T4 engagement on cancer cells is expected to enhance immune responses within the tumor microenvironment while minimizing the risk of peripheral toxicity. Furthermore, by restricting the antibody to tumor cells expressing 5T4, the 5T4xCD40 bispecific antibody is expected to overcome antigenic silencing resulting from widespread peripheral CD40 expression, thereby allowing the molecule to accumulate within the tumor.

[0019] According to one embodiment of the present disclosure, a multispecific antibody is provided, comprising a first antibody or antigen-binding fragment having binding specificity for CD40 protein, and a second antibody or antigen-binding fragment having binding specificity for 5T4 protein, wherein the multispecific antibody more effectively activates CD40 on target cells expressing 5T4 protein than on reference cells that do not express 5T4 protein, or wherein the multispecific antibody does not activate CD40 on reference cells that do not express 5T4 protein.

[0020] In some embodiments, the multispecific antibody activates CD40 on target cells expressing the 5T4 protein at least 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold greater than CD40 on reference cells that do not express the 5T4 protein. In some embodiments, the activation is measured at a multispecific antibody concentration of 0.001 nM to 200 nM, preferably 0.1 nM to 100 nM. In some embodiments, the activation is measured using a panel of CD40 functional assays; preferably, the activation is measured using an NFκB reporter gene assay, an IL-12 secretion assay, a CD80 expression assay, a CD86 expression assay, a Ki67 expression assay, or a Ki67 / CD86 expression assay.

[0021] In some embodiments, the first antibody or antigen-binding fragment comprises two or three tandem single domain (VHH) anti-CD40 antibodies. In some embodiments, the second antibody or antigen-binding fragment comprises a conventional VH / VL Fab fragment. In some embodiments, the two or three tandem VHH anti-CD40 antibodies and the conventional VH / VL Fab fragment are each fused to the N-terminus of each of the two chains of the Fc fragment.

[0022] In some embodiments, the first antibody or antigen-binding fragment comprises two separate (VHH) anti-CD40 antibodies, each fused to the C-terminus of each of the two chains of the Fc fragment, and wherein the second antibody or antigen-binding fragment comprises two conventional VH / VL Fab fragments, each fused to the N-terminus of each of the two chains of the Fc fragment.

[0023] In some embodiments, the Fc fragment is a human IgG1, IgG2, or IgG4 fragment.In some embodiments, the Fc fragment comprises the substitutions L234A, L235A and N297A, L234A and L235A, or N297A according to Kabat numbering.

[0024] In some embodiments, the first antibody or antigen-binding fragment comprises one or more single-domain (VHH) anti-CD40 antibodies, each of which comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 14, a CDR2 comprising the amino acid sequence of SEQ ID NO: 15, 63, or 64, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 5. In some embodiments, each of these VHH antibodies comprises an amino acid sequence selected from SEQ ID NO: 1 and 53-62. In some embodiments, each of these VHH antibodies comprises the amino acid sequence of SEQ ID NO: 54.

[0025] In some embodiments, the second antibody or antigen-binding fragment competes for binding to 5T4 protein with antibody 14G12 or 159D5, wherein the antibody 14G12 comprises the VH of SEQ ID NO:73 and the VL of SEQ ID NO:74, and the antibody 159D5 comprises the VH of SEQ ID NO:121 and the VL of SEQ ID NO:122.

[0026] In some embodiments, the second antibody or antigen-binding fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region comprising VH CDR1, VH CDR2 and VH CDR3, the light chain variable region comprising VL CDR1, VLCDR2 and VL CDR3, wherein the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 respectively comprise the amino acid sequence of SEQ ID NO: 130-80. In some embodiments, the VH comprises an amino acid sequence selected from SEQ ID NO: 73 and 81-90, and the VL comprises an amino acid sequence selected from SEQ ID NO: 74 and 91-100. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 83 or 89, and the VL comprises the amino acid sequence of SEQ ID NO: 91.

[0027] In some embodiments, the second antibody or antigen-binding fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region comprising VH CDR1, VH CDR2 and VH CDR3, the light chain variable region comprising VL CDR1, VLCDR2 and VL CDR3, wherein the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 respectively comprise the amino acid sequence of SEQ ID NO: 103-108. In some embodiments, the VH comprises an amino acid sequence selected from SEQ ID NO: 101 and 109-115, and the VL comprises an amino acid sequence selected from SEQ ID NO: 102 and 116-120. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 113, and the VL comprises the amino acid sequence of SEQ ID NO: 120.

[0028] In some embodiments, the second antibody or antigen-binding fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region comprising VH CDR1, VH CDR2 and VH CDR3, the light chain variable region comprising VL CDR1, VLCDR2 and VL CDR3, wherein the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 respectively comprise the amino acid sequence of SEQ ID NO: 123-128. In some embodiments, the VH comprises an amino acid sequence selected from SEQ ID NO: 121 and 129-131, and the VL comprises an amino acid sequence selected from SEQ ID NO: 122 and 132-137. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 130, and the VL comprises the amino acid sequence of SEQ ID NO: 133.

[0029] Also provided are compositions, methods, and uses for treating a disease. In one embodiment, they are used to treat cancer in a patient in need thereof. In some embodiments, the treatment further comprises administering an immune checkpoint inhibitor. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The binding activities of the anti-CD40 monospecific antibodies to the human CD40 antigen and the cynomolgus monkey CD40 antigen are shown.

[0031] Figure 2 The binding activity of anti-CD40 monospecific antibodies to Jurkat cells overexpressing human CD40 is shown.

[0032] Figure 3ELISA binding results of anti-CD40 antibodies to human OX40 or human 4-1BB are presented.

[0033] Figure 4 The format of the Claudin 18.2 / CD40 bispecific antibody is shown.

[0034] Figure 5 The anti-Claudin 18.2 / CD40 bispecific antibody was shown to bind to human dendritic cells with activity weaker than that of the benchmark antibody, selenulumab.

[0035] Figure 6 The anti-Claudin 18.2 / CD40 bispecific antibody was shown to bind to human B cells with activity weaker than that of the benchmark antibody, Selumetumab.

[0036] Figure 7 CD40 monospecific antibodies were shown to activate CD40 signaling with much lower activity than the benchmark antibody selumetumab.

[0037] Figure 8 The anti-Claudin 18.2 / CD40 bispecific antibody was shown to activate CD40 signaling in a Claudin 18.2-dependent manner.

[0038] Figure 9 The results showed that the anti-claudin 18.2 / CD40 bispecific antibody activated dendritic cells to secrete IL-12 in a claudin 18.2-dependent manner.

[0039] Figure 10 The anti-Claudin 18.2 / CD40 bispecific antibody activates dendritic cells to express CD80 (A) and CD86 (B) in a Claudin 18.2-dependent manner.

[0040] Figure 11 Anti-Claudin 18.2 / CD40 bispecific antibody is shown to increase B cell proliferation (A) and activation (B) in a Claudin 18.2-dependent manner.

[0041] Figure 12 The binding activity of the humanized 2p442 antibody to Jurkat cells overexpressing human CD40 is shown.

[0042] Figure 13 The results showed that the humanized anti-claudin 18.2 / CD40 bispecific antibody had CD40 activation activity comparable to that of its chimeric antibody.

[0043] Figure 14 The results showed that the humanized anti-claudin 18.2 / CD40 bispecific antibody has the function of inducing IL-12 secretion comparable to its chimeric antibody.

[0044] Figure 15 The humanized anti-Claudin 18.2 / CD40 bispecific antibody showed that it had the ability to induce the expression of CD80 (A) and CD86 (B) on dendritic cells, comparable to its chimeric antibody.

[0045] Figure 16 The humanized anti-Claudin 18.2 / CD40 bispecific antibody showed comparable B cell proliferation (A) and activation (B) induction functions to its chimeric antibody.

[0046] Figure 17 In vivo tumor growth inhibition by anti-Claudin 18.2 / CD40 chimeric bispecific antibody is shown.

[0047] Figure 18 Shown are the in vivo study design (A) and blood biochemistry analysis (B), tumor infiltration immunophenotyping (IPT) analysis (CF), and spleen IPT analysis (GI) of the anti-Claudin 18.2 / CD40 chimeric bispecific antibody.

[0048] Figure 19 It was shown that the anti-5T4 / CD40 bispecific antibody with the "2+2b11" format activated CD40 signaling in a 5T4-dependent manner.

[0049] Figure 20 The anti-5T4 / CD40 bispecific antibody was shown to activate dendritic cells to secrete IL-12 in a 5T4-dependent manner.

[0050] Figure 21 Anti-5T4 / CD40 bispecific antibody activates dendritic cells to express CD80 (A) and CD86 (B) in a 5T4-dependent manner.

[0051] Figure 22 Shown are the "2+2b11" format, "1+1b12" format, "2+2b13" format, "1+2b16" format, "1+2b17" format, and "1+3b18" format of the anti-5T4 / CD40 bispecific antibody.

[0052] Figure 23 The binding activity of the anti-5T4 / CD40 bispecific antibody to Jurkat cells overexpressing human CD40 is shown.

[0053] Figure 24 Anti-5T4 / CD40 bispecific antibodies with different formats were shown to activate CD40 signaling with varying potency.

[0054] Figure 25Anti-5T4 / CD40 bispecific antibodies with different formats were shown to activate dendritic cells to secrete IL-12 with varying potency.

[0055] Figure 26 In vivo tumor growth inhibition by anti-5T4 / CD40 bispecific antibodies is shown.

[0056] Figure 27 Shown are ex vivo blood immunophenotyping (IPT) analyses of the tested anti-5T4 / CD40 bispecific antibodies.

[0057] Figure 28 Shown is an ex vivo tumor infiltration IPT analysis of the tested anti-5T4 / CD40 bispecific antibodies.

[0058] Figure 29 The results showed that the humanized anti-5T4 / CD40 bispecific antibody b16(42p155z2)-LALA had comparable binding activity to human dendritic cells as its chimeric antibody.

[0059] Figure 30 The results showed that the humanized anti-5T4 / CD40 bispecific antibody b16(42p155z2)-LALA had CD40 activation activity comparable to that of its chimeric antibody.

[0060] Figure 31 The results showed that the humanized anti-5T4 / CD40 bispecific antibody b16(42p155z2)-LALA has the function of inducing IL-12 secretion comparable to its chimeric antibody. DETAILED DESCRIPTION

[0061] definition

[0062] It should be noted that the term "a" or "an" entity refers to one or more entities in that entity; for example, "an antibody" should be understood to mean one or more antibodies. Therefore, the terms "a" (or "an"), "one or more" and "at least one" are used interchangeably herein.

[0063] By "sequence identity" of a polynucleotide or polynucleotide region (or polypeptide or polypeptide region) to another sequence is meant that, when aligned, the percentage of bases (or amino acids) that are the same when the two sequences are compared is the same. Such alignments and homology or sequence identity percentages can be determined using software programs known in the art, such as those described in "Current Protocols in Molecular Biology" by Ausubel et al., eds. (2007). Preferably, the alignment is performed using default parameters. One alignment program is BLAST, using default parameters. In particular, the programs are BLASTN and BLASTP, using the following default parameters: Genetic code = standard; filter = none; strand = both; cutoff = 60; expectation = 10; Matrix = BLOSUM62; Descriptions = 50 sequences; sort by = HIGH SCORE; Databases = non-redundant; GenBank + EMBL + DDBJ + PDB + GenBank CDS translations + SwissProtein + SPupdate + PIR. Biologically equivalent polynucleotides are polynucleotides having the above specified percentage homology and encoding polypeptides having the same or similar biological activity.

[0064] The term "equivalent nucleic acid or polynucleotide" refers to a nucleic acid having a nucleotide sequence that has a certain degree of homology or sequence identity with the nucleotide sequence of the nucleic acid or its complement. The homologue of a double-stranded nucleic acid is intended to include a nucleic acid having a nucleotide sequence that has a certain degree of homology with it or its complement. On the one hand, the homologue of a nucleic acid can hybridize with a nucleic acid or its complement. Similarly, an "equivalent polypeptide" refers to a polypeptide that has a certain degree of homology or sequence identity with the amino acid sequence of a reference polypeptide. In some aspects, the sequence identity is at least about 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%. In some aspects, compared with a reference polypeptide or polynucleotide, an equivalent polypeptide or polynucleotide has one, two, three, four or five additions, deletions, substitutions and combinations thereof. In some aspects, an equivalent sequence retains the activity (e.g., epitope binding) or structure (e.g., salt bridge) of a reference sequence.

[0065] As used herein, "antibody" or "antigen-binding polypeptide" refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. An antibody can be a complete antibody and any antigen-binding fragment or single chain thereof. Thus, the term "antibody" includes any protein or peptide containing molecule that comprises at least a portion of an immunoglobulin molecule that has biological activity of binding to an antigen. Examples include, but are not limited to, a heavy or light chain complementary determining region (CDR) or ligand binding portion thereof, a heavy or light chain variable region, a heavy or light chain constant region, a framework (FR) region, or any portion thereof, or at least a portion of a binding protein.

[0066] Single domain antibodies (sdAb), also known as nano antibodies, are antibody fragments consisting of a single monomer variable antibody domain. Nano antibodies produced by camelids and certain other animals are also called VHH fragments. Like complete antibodies, nano antibodies can selectively bind to specific antigens. Due to the molecular weight of only 12kDa to 15kDa, single domain antibodies are much smaller than ordinary antibodies (150kDa to 160kDa). Single domain antibodies, due to their small size and single chain properties, can be particularly suitable as fragments contained in other proteins (such as chimeric antigen receptors (CAR) and bispecific antibodies).

[0067] As used herein, the term "antibody fragment" or "antigen-binding fragment" is a portion of an antibody, such as F(ab')2, F(ab)2, Fab', Fab, Fv, scFv, etc. Regardless of the structure, an antibody fragment binds to the same antigen recognized by the intact antibody. The term "antibody fragment" includes aptamers, spiegelmers, and diabodies. The term "antibody fragment" also includes any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex.

[0068] The antibodies, antigen-binding polypeptides, variants or derivatives thereof disclosed herein include, but are not limited to, polyclonal antibodies, monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, primatized antibodies or chimeric antibodies, single-chain antibodies, epitope-binding fragments (e.g., Fab, Fab' and F(ab')2, Fd, Fvs, single-chain Fvs (scFv)), single-chain antibodies, disulfide-linked Fvs (sdFv), fragments comprising a VK or VH domain, fragments produced by a Fab expression library, and anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies to the LIGHT antibodies disclosed herein). The immunoglobulin or antibody molecules disclosed herein can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecules.

[0069] "Specific binding" or "specific for..." generally means that the antibody binds to the epitope via its antigen binding domain, and that this binding requires some complementarity between the antigen binding domain and the epitope. According to this definition, an antibody is said to "specifically bind" to an epitope when it binds to the epitope via its antigen binding domain more easily than it binds to a random, unrelated epitope. The term "specificity" is used herein to qualify the relative affinity with which an antibody binds to a certain epitope. For example, antibody "A" can be said to have a higher specificity for a given epitope than antibody "B", or antibody "A" can be said to bind to epitope "C" with a higher specificity than to a related epitope "D".

[0070] As used herein, the term "treatment" refers to both therapeutic treatment and preventive or defensive measures, wherein the goal is to prevent or slow down (mitigate) undesirable physiological changes or conditions, such as cancer progression. Beneficial or desired clinical results include, but are not limited to, the alleviation of detectable or undetectable symptoms, the weakening of the degree of disease, the stable (i.e., non-exacerbated) state of the disease, the delay or slowing of disease progression, the improvement or alleviation of the disease state, and (partial or complete) relief. "Treatment" can also refer to a prolonged survival period compared to the expected survival period when not receiving treatment. Individuals requiring treatment include individuals who have suffered from a condition or condition and individuals who are susceptible to a condition or condition or individuals to prevent a condition or condition.

[0071] "Subject" or "individual" or "animal" or "patient" or "mammal" means any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or treatment is desired. Mammalian subjects include humans, domesticated animals, farm animals, and zoo, sport, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, and the like.

[0072] As used herein, phrases such as "a patient in need of treatment" or "a subject in need of treatment" include subjects, such as mammalian subjects, who would benefit from administration of an antibody or composition of the disclosure for, eg, detection, diagnostic procedures, and / or therapy.

[0073] Tumor antigen-dependent CD40 agonist antibodies for improving therapeutic index

[0074] CD40 is widely expressed in hematopoietic and non-hematopoietic tissues. CD40 regulates immunity and therefore provides a potential pathway for cancer immunotherapy. It has been shown that activation of CD40 can enable DCs to drive CD8 T cell responses. In addition, CD40 activation achieves immune activation that is independent of innate immune receptors such as stimulator of interferon genes (STING) or Toll-like receptors (TLRs). Therefore, a lot of work has been done to develop CD40 agonist antibodies for the treatment of cancer.

[0075] However, unlike inhibitory antibodies, agonist approaches present significant challenges regarding dosage and schedule, which complicate drug development. Another complicating factor is that CD40 agonist antibodies are often associated with moderate to severe toxicities such as cytokine release syndrome (CRS). This may be attributed to CD40 activation at non-cancerous sites. The current solution is to treat these toxicities as they occur, which is difficult to manage, expensive, and prohibitive for patients.

[0076] Through careful design and screening, the inventors were unexpectedly able to identify CD40 agonist antibodies with different CD40 activation activities in the presence or absence of tumor-associated antigens (TAAs). In particular, a long list of novel CD40 antibodies was identified, all of which had significantly reduced CD40 agonist activity compared to the reference antibody selukumab, the most extensively studied CD40 antibody candidate (see, e.g., Examples 4 and 5). Figure 8 A). However, when used in a bispecific or multispecific format also containing an anti-TAA unit, these antibodies exhibited CD40 activation activity far greater than that of selumetumab (see e.g. Figure 8 B. Figures 9 to 11 ).

[0077] These antibodies can therefore be referred to as "TAA-dependent CD40 agonist antibodies." These TAA-dependent CD40 agonist antibodies have shown greatly improved anti-tumor efficacy in animal models (see, e.g., Examples 9 and Figure 16 ). It is also inevitable that they will result in greatly reduced toxicity because they do not induce CD40 activity in tissues or organs that do not express the targeted TAA.

[0078] More interestingly, these newly identified CD40 agonist antibodies were further classified into four classes. Figure 8 As shown and summarized in Table 5, the tier 4 antibodies did not activate CD40 in the absence of TAAs and had only relatively weak CD40 activation in the presence of TAAs; the tier 3 antibodies had moderate CD40 activation in the absence of TAAs and had the most potent CD40 activation in the presence of TAAs; and the tier 2 antibodies had marginal CD40 activation in the absence of TAAs and moderate CD40 activation in the presence of TAAs.

[0079] Class 1 antibodies are of the greatest interest. They have low or no CD40 activation in the absence of TAAs and potent CD40 activation in the presence of TAAs on target cells. Class 1 antibodies include 42p155, 2p834, 2p931, 42p655, and 2p1294. These antibodies are believed to have the highest therapeutic index and, therefore, the greatest clinical potential.

[0080] Thus, in one embodiment of the present disclosure, a TAA-dependent CD40 agonist antibody is provided. A TAA-dependent CD40 agonist antibody is an antibody that, when presented in a bispecific or multispecific antibody format that also includes an anti-TAA unit, activates CD40 only on cells expressing the TAA and activates more CD40 on reference cells that lack the TAA. For a fair comparison, in some embodiments, the reference cells differ from the TAA-expressing cells only in the expression of the TAA.

[0081] In some embodiments, the difference in CD40 activation between cells expressing the TAA and cells lacking the TAA is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 21-fold, 22-fold, 23-fold, 24-fold, 25-fold, 26-fold, 27-fold, 28-fold, 29-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, or 100-fold.

[0082] In some embodiments, the TAA-dependent CD40 agonist antibody activates CD40 less than selukumab in the absence of a TAA, such as 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% less than selukumab.

[0083] In some embodiments, the TAA on the TAA-expressing cells is at least detectable by conventional methods, such as immunohistochemical staining. In some embodiments, the TAA expression is at least at the average level of TAA-expressing tumors.

[0084] In some embodiments, activation measurements are performed using a bispecific or multispecific antibody present at a concentration of 0.001 nM to 1000 nM. In some embodiments, the antibody concentration is 0.01 nM to 500 nM. In some embodiments, the antibody concentration is 0.1 nM to 200 nM. In some embodiments, the antibody concentration is 0.1 nM to 20 nM. In some embodiments, the antibody concentration is 1 nM to 20 nM. In some embodiments, the antibody concentration is about 0.001 nM, 0.01 nM, 0.1 nM, 0.14 nM, 1 nM, 1.2 nM, 11 nM, or 100 nM.

[0085] Different assays for measuring CD40 activation are available, including the use of commercially available kits. In one example, the target cells are CHO cells, and the activation is measured using an NFκB reporter gene assay. In another example, the target cells are dendritic cells (DCs), and the CD40 activation is measured using IL-12 secretion, CD80, and CD86 expression. In yet another example, the target cells are B cells, and the CD40 activation is measured using Ki67 and / or CD86 expression.

[0086] Also provided are exemplary TAA-dependent CD40 agonist antibodies that are as long as proteins (e.g., multispecific antibodies, chimeric antigen receptors (CARs)). In one embodiment of the present disclosure, single-domain antibodies and polypeptides comprising such single-domain antibodies are provided. In one embodiment of the present disclosure, single-domain antibodies or polypeptides comprising the single-domain antibodies are provided, wherein the single-domain antibodies comprise CDR1, CDR2, and CDR3, each having the CDR1, CDR2, and CDR3 sequences of antibody 42p155 (SEQ ID NO: 1). In some embodiments, the CDR1, CDR2, and CDR3 comprise the amino acid sequences of SEQ ID NOs: 14-16, respectively.

[0087] Analysis has shown that certain residues in the CDR2 of 42p155 may undergo post-translational modification (PTM). Therefore, mutations are made to prevent such PTMs (hence the term PTM de-risking form), including NG=>NA or QG. See, for example, SEQ ID NOs: 63 and 64. Thus, in some embodiments, the CDR1, CDR2, and CDR3 comprise the amino acid sequences of SEQ ID NOs: 14, 63, and 16, respectively. Thus, in some embodiments, the CDR1, CDR2, and CDR3 comprise the amino acid sequences of SEQ ID NOs: 14, 64, and 16, respectively.

[0088] In some embodiments, humanized forms of 42p155 and their PTM-derisked counterparts are also provided, such as those provided in SEQ ID NOs: 53-62. In some embodiments, the humanized antibodies include a back mutation selected from 1P, 2S, 88P, and 98Q according to Kabat numbering. In some embodiments, the humanized antibodies include a back mutation 98Q. In some embodiments, the humanized antibodies include back mutations 88P and 98Q. In some embodiments, the humanized antibodies include back mutations 1P, 2S, 88P, and 98Q.

[0089] In some embodiments, in a humanized form, CDR1 comprises the amino acid sequence of SEQ ID NO: 14, CDR2 comprises the amino acid sequence of SEQ ID NO: 15, and CDR3 comprises the amino acid sequence of SEQ ID NO: 16. In some embodiments, the antibody or polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 53, 54, 57, and 60. In some embodiments, the antibody comprises the recited CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NOs: 53, 54, 57, or 60.

[0090] In some embodiments, in a humanized form, CDR1 comprises the amino acid sequence of SEQ ID NO: 14, CDR2 comprises the amino acid sequence of SEQ ID NO: 63, and CDR3 comprises the amino acid sequence of SEQ ID NO: 16. In some embodiments, the antibody or polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 55, 58, and 61. In some embodiments, the antibody comprises the recited CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NOs: 55, 58, or 61.

[0091] In some embodiments, in a humanized form, CDR1 comprises the amino acid sequence of SEQ ID NO: 14, CDR2 comprises the amino acid sequence of SEQ ID NO: 64, and CDR3 comprises the amino acid sequence of SEQ ID NO: 16. In some embodiments, the antibody or polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 56, 59, and 62. In some embodiments, the antibody comprises the recited CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NOs: 56, 59, or 62.

[0092] In another embodiment, a single-domain antibody or a polypeptide comprising the single-domain antibody is provided, wherein the single-domain antibody comprises CDR1, CDR2, and CDR3, which respectively have the CDR1, CDR2, and CDR3 sequences of antibody 2p834 (SEQ ID NO: 2). In some embodiments, the CDR1, CDR2, and CDR3 respectively comprise the amino acid sequences of SEQ ID NOs: 17-19.

[0093] In some embodiments, humanized forms of 2p834 are also provided, such as those provided in SEQ ID NOs: 65-68. In some embodiments, the humanized antibody comprises a back mutation selected from 1P, 2S, 88P, and 98Q according to Kabat numbering. In some embodiments, the humanized antibody comprises a back mutation 98Q. In some embodiments, the humanized antibody comprises a back mutation 88P and 98Q. In some embodiments, the humanized antibody comprises a back mutation 1P, 2S, 88P, and 98Q. In some embodiments, the antibody comprises the listed CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NOs: 1, 65, 66, 67, or 68.

[0094] In another embodiment, a single domain antibody or a polypeptide comprising the single domain antibody is provided, wherein the single domain antibody comprises CDR1, CDR2 and CDR3, which respectively have the CDR1, CDR2 and CDR3 sequences of antibody 2p931 (SEQ ID NO: 3). In some embodiments, the CDR1, CDR2 and CDR3 respectively comprise the amino acid sequences of SEQ ID NOs: 20-22. In some embodiments, the antibody comprises the listed CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 3.

[0095] In another embodiment, a single domain antibody or a polypeptide comprising the single domain antibody is provided, wherein the single domain antibody comprises CDR1, CDR2 and CDR3, which respectively have the CDR1, CDR2 and CDR3 sequences of antibody 42p655 (SEQ ID NO: 4). In some embodiments, the CDR1, CDR2 and CDR3 respectively comprise the amino acid sequences of SEQ ID NOs: 23-25. In some embodiments, the antibody comprises the listed CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 4.

[0096] In another embodiment, a single domain antibody or a polypeptide comprising the single domain antibody is provided, wherein the single domain antibody comprises CDR1, CDR2 and CDR3, which respectively have the CDR1, CDR2 and CDR3 sequences of antibody 2p1294 (SEQ ID NO: 5). In some embodiments, the CDR1, CDR2 and CDR3 respectively comprise the amino acid sequences of SEQ ID NO: 26-28. In some embodiments, the antibody comprises the listed CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 5.

[0097] In another embodiment, a single domain antibody or a polypeptide comprising the single domain antibody is provided, wherein the single domain antibody comprises CDR1, CDR2 and CDR3, which respectively have the CDR1, CDR2 and CDR3 sequences of antibody 2p957 (SEQ ID NO: 6). In some embodiments, the CDR1, CDR2 and CDR3 respectively comprise the amino acid sequences of SEQ ID NO: 29-31. In some embodiments, the antibody comprises the listed CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 6.

[0098] In another embodiment, a single domain antibody or a polypeptide comprising the single domain antibody is provided, wherein the single domain antibody comprises CDR1, CDR2 and CDR3, which respectively have the CDR1, CDR2 and CDR3 sequences of antibody 42p495 (SEQ ID NO: 7). In some embodiments, the CDR1, CDR2 and CDR3 respectively comprise the amino acid sequences of SEQ ID NOs: 32-34. In some embodiments, the antibody comprises the listed CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 7.

[0099] In another embodiment, a single domain antibody or a polypeptide comprising the single domain antibody is provided, wherein the single domain antibody comprises CDR1, CDR2 and CDR3, which respectively have the CDR1, CDR2 and CDR3 sequences of antibody 3p78 (SEQ ID NO: 8). In some embodiments, the CDR1, CDR2 and CDR3 respectively comprise the amino acid sequences of SEQ ID NO: 35-37. In some embodiments, the antibody comprises the listed CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 8.

[0100] In another embodiment, a single domain antibody or a polypeptide comprising the single domain antibody is provided, wherein the single domain antibody comprises CDR1, CDR2 and CDR3, which respectively have the CDR1, CDR2 and CDR3 sequences of antibody 2p415 (SEQ ID NO: 9). In some embodiments, the CDR1, CDR2 and CDR3 respectively comprise the amino acid sequences of SEQ ID NO: 38-40. In some embodiments, the antibody comprises the listed CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 9.

[0101] In another embodiment, a single domain antibody or a polypeptide comprising the single domain antibody is provided, wherein the single domain antibody comprises CDR1, CDR2 and CDR3, which respectively have the CDR1, CDR2 and CDR3 sequences of antibody 2p442 (SEQ ID NO: 10). In some embodiments, the CDR1, CDR2 and CDR3 respectively comprise the amino acid sequences of SEQ ID NOs: 41-43. In some embodiments, the antibody comprises the listed CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 10.

[0102] In some embodiments, humanized forms of 2p442 are also provided, such as those provided in SEQ ID NOs: 69-72. In some embodiments, the antibodies comprise the listed CDR1, CDR2, and CDR3 and have at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NOs: 10, 69, 70, 71, or 72.

[0103] In another embodiment, a single domain antibody or a polypeptide comprising the single domain antibody is provided, wherein the single domain antibody comprises CDR1, CDR2 and CDR3, which respectively have the CDR1, CDR2 and CDR3 sequences of antibody 2p551 (SEQ ID NO: 11). In some embodiments, the CDR1, CDR2 and CDR3 respectively comprise the amino acid sequences of SEQ ID NO: 44-46. In some embodiments, the antibody comprises the listed CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 11.

[0104] In another embodiment, a single domain antibody or a polypeptide comprising the single domain antibody is provided, wherein the single domain antibody comprises CDR1, CDR2 and CDR3, which respectively have the CDR1, CDR2 and CDR3 sequences of antibody 2p80 (SEQ ID NO: 12). In some embodiments, the CDR1, CDR2 and CDR3 respectively comprise the amino acid sequences of SEQ ID NOs: 47-49. In some embodiments, the antibody comprises the listed CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 12.

[0105] In another embodiment, a single domain antibody or a polypeptide comprising the single domain antibody is provided, wherein the single domain antibody comprises CDR1, CDR2 and CDR3, which respectively have the CDR1, CDR2 and CDR3 sequences of antibody 2p1130 (SEQ ID NO: 13). In some embodiments, the CDR1, CDR2 and CDR3 respectively comprise the amino acid sequences of SEQ ID NOs: 50-52. In some embodiments, the antibody comprises the listed CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 14.

[0106] In some embodiments, anti-CD40 antibodies and antigen-binding fragments are also provided that compete with any of the antibodies of the present disclosure for binding to human CD40. In some embodiments, anti-CD40 antibodies and antigen-binding fragments are also provided that bind to the same epitope as any of the antibodies of the present disclosure. In some embodiments, anti-CD40 antibodies and antigen-binding fragments are also provided that comprise the CDR1, CDR2, and CDR3 of an antibody of the present disclosure.

[0107] Also provided is a composition comprising the antibody or the polypeptide and a pharmaceutically acceptable carrier.

[0108] It will also be understood by those of ordinary skill in the art that the antibodies disclosed herein can be modified so that they differ in amino acid sequence from the naturally occurring binding polypeptides from which they are derived. For example, a polypeptide or amino acid sequence derived from a specified protein can be similar, for example, having a certain percentage of identity with the starting sequence, for example, it can be 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identical to the starting sequence. In some embodiments, the modified antibodies or fragments retain the specified CDR sequences.

[0109] In certain embodiments, the antibodies include amino acid sequences or one or more moieties that are not normally associated with antibodies. Exemplary modifications are described in more detail below. For example, the antibodies of the present disclosure may include flexible linker sequences or may be modified to add functional moieties (e.g., PEG, drugs, toxins, or labels).

[0110] Bispecific and multispecific antibodies

[0111] As provided, the CD40 agonist antibodies disclosed herein are particularly useful for preparing bispecific and multispecific antibodies due at least in part to the enhanced therapeutic index of these antibodies and their small size.

[0112] CD40 is widely expressed in hematopoietic and non-hematopoietic tissues. CD40 regulates immunity and therefore provides a potential pathway for cancer immunotherapy. It has been shown that activation of CD40 can enable DCs to drive CD8 T cell responses. In addition, CD40 activation achieves immune activation that is independent of innate immune receptors such as stimulator of interferon genes (STING) or Toll-like receptors (TLRs). Therefore, a lot of work has been done to develop CD40 agonist antibodies for the treatment of cancer.

[0113] However, unlike inhibitory antibodies, agonist approaches present significant challenges regarding dosage and schedule, which complicate drug development. Another complicating factor is that CD40 agonist antibodies are often associated with moderate to severe toxicities such as cytokine release syndrome (CRS). This may be attributed to CD40 activation at non-cancerous sites. The current solution is to treat these toxicities as they occur, which is difficult to manage, expensive, and prohibitive for patients.

[0114] Thus, in one embodiment, a bispecific antibody is provided that comprises a TAA-dependent CD40 agonist antibody or antigen-binding fragment thereof, and a second antibody or antigen-binding fragment having binding specificity for a non-CD40 target antigen. In some embodiments, a third or fourth specificity is further included. In some embodiments, the non-CD40 target antigen is a tumor antigen.

[0115] A TAA-dependent CD40 agonist antibody is an antibody that, when presented in such a bispecific or multispecific antibody format that also comprises an anti-TAA unit, activates CD40 on cells expressing the TAA more than it activates CD40 on reference cells lacking the TAA. For a fair comparison, in some embodiments, the reference cells differ from the TAA-expressing cells only in the expression of the TAA.

[0116] In some embodiments, the difference in CD40 activation between cells expressing the TAA and cells lacking the TAA is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 21-fold, 22-fold, 23-fold, 24-fold, 25-fold, 26-fold, 27-fold, 28-fold, 29-fold, 30-fold, 35-fold, 40-fold, 45-fold, or 50-fold.

[0117] In some embodiments, the TAA-dependent CD40 agonist antibody activates CD40 less than selukumab in the absence of a TAA, such as 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% less than selukumab.

[0118] In some embodiments, the TAA on the TAA-expressing cells is at least detectable by conventional methods, such as immunohistochemical staining. In some embodiments, the TAA expression is at least at the average level of TAA-expressing tumors.

[0119] In some embodiments, activation measurements are performed using a bispecific or multispecific antibody present at a concentration of 0.001 nM to 1000 nM. In some embodiments, the antibody concentration is 0.01 nM to 500 nM. In some embodiments, the antibody concentration is 0.1 nM to 200 nM. In some embodiments, the antibody concentration is 0.1 nM to 20 nM. In some embodiments, the antibody concentration is 1 nM to 20 nM. In some embodiments, the antibody concentration is about 0.001 nM, 0.01 nM, 0.1 nM, 0.14 nM, 1 nM, 1.2 nM, 11 nM, or 100 nM.

[0120] Different assays for measuring CD40 activation are available, including the use of commercially available kits. In one example, the target cells are CHO cells, and the activation is measured using an NFκB reporter gene assay. In another example, the target cells are dendritic cells (DCs), and the CD40 activation is measured using IL-12 secretion, CD80, or CD86 expression. In yet another example, the target cells are B cells, and the CD40 activation is measured using Ki67 and / or CD86 expression.

[0121] In some embodiments, the TAA-dependent CD40 agonist antibodies are as disclosed in the preceding sections, such as 42p155, 2p834, 2p931, 42p655, 2p1294, 2p957, 42p495, 3p78, 2p415, 2p442, 2p551, 2p80, and 2p1130, and biological equivalents thereof.

[0122] The abundance of tumor antigens is known in the art, and new tumor antigens can be easily identified by screening. Non-limiting examples of tumor antigens include claudin 18.2, EGFR, Her2, EpCAM, CD20, CD30, CD33, CD47, CD52, CD133, CD73, CEA, gpA33, mucin, TAG-72, CIX, PSMA, folate binding protein, GD2, GD3, GM2, VEGF, VEGFR, integrin, αVβ3, α5β1, ERBB2, ERBB3, MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP and tenascin. In some embodiments, the bispecific antibody is specific for CD40 and claudin 18.2.

[0123] The present inventors designed a panel of 5T4 x CD40 bispecific antibodies with anti-5T4 / anti-CD40 moieties of varying properties and in different formats. These bispecific antibodies were evaluated in CD40 reporter cells and co-cultured with 5T4-expressing target cells. Their efficacy was further confirmed in vitro by measuring IL12 production by monocyte-derived dendritic cells (DCs) and CD80 and CD86 expression on DCs and B cells. Furthermore, in vivo anti-tumor efficacy was determined in CD40 humanized C57BL / 6 mice bearing MC38-hu5T4 tumors.

[0124] It has been observed that bispecific antibodies that activate CD40 signaling in a 5T4-dependent manner show the best in vitro and in vivo performance. In addition, multiple formats of bispecific antibodies were tested, and two of them (b16 and b18) showed superiority in inducing more potent CD40 agonism in a 5T4-dependent manner.

[0125] In vivo testing with 5T4 x CD40 demonstrated potent antitumor efficacy, significantly exceeding clinical benchmarks at similar doses. Furthermore, the resulting tumor-free mice were resistant to tumor rechallenge, demonstrating the establishment of a long-lasting memory response. Furthermore, ex vivo analysis demonstrated focal immune activation in the tumor without peripheral activation, confirming the safety of these bispecific antibodies.

[0126] A. Bispecific Antibodies with 5T4-Dependent Anti-CD40 Portions

[0127] According to one embodiment of the present disclosure, a bispecific antibody or a multispecific antibody incorporating the bispecific antibody is provided, which comprises an anti-5T4 portion and an anti-CD40 portion. In some embodiments, the anti-CD40 portion comprises one, two, three, or four anti-CD40 antibodies or fragments having 5T4-dependent agonist activity.

[0128] The present inventors have prepared and tested single-domain anti-CD40 antibodies that have significantly reduced CD40 agonist activity compared to the reference antibody selukumab (the most extensively studied CD40 antibody candidate). When used in a bispecific or multispecific format that also contains an anti-tumor-associated antigen (TAA, such as 5T4) unit, these antibodies exhibit CD40 activation activity far greater than that of selukumab. These antibodies can therefore be referred to as "5T4-dependent CD40 agonist antibodies." These 5T4-dependent CD40 agonist antibodies have shown greatly improved anti-tumor efficacy in animal models. It is also inevitable that they will result in greatly reduced toxicity because they do not induce CD40 activity in tissues or organs that do not express 5T4.

[0129] A 5T4-dependent CD40 agonist antibody is an antibody that, when presented in a bispecific or multispecific antibody format that also comprises an anti-5T4 unit, activates CD40 only on cells expressing 5T4 and activates more CD40 on reference cells lacking 5T4. For a fair comparison, in some embodiments, the reference cells differ from the 5T4-expressing cells only in the expression of the TAA.

[0130] In some embodiments, the difference in CD40 activation between cells expressing 5T4 and cells lacking 5T4 is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 21-fold, 22-fold, 23-fold, 24-fold, 25-fold, 26-fold, 27-fold, 28-fold, 29-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, or 100-fold.

[0131] In some embodiments, the 5T4-dependent CD40 agonist antibody activates CD40 less than selukumab in the absence of 5T4, such as 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% less than selukumab.

[0132] In some embodiments, 5T4 on cells expressing 5T4 is at least detectable by conventional methods, such as immunohistochemical staining. In some embodiments, the 5T4 expression is at least at the average level of tumors expressing 5T4.

[0133] In some embodiments, activation measurements are performed using a bispecific or multispecific antibody present at a concentration of 0.001 nM to 1000 nM. In some embodiments, the antibody concentration is 0.01 nM to 500 nM. In some embodiments, the antibody concentration is 0.1 nM to 200 nM. In some embodiments, the antibody concentration is 0.1 nM to 20 nM. In some embodiments, the antibody concentration is 1 nM to 20 nM. In some embodiments, the antibody concentration is about 0.001 nM, 0.01 nM, 0.1 nM, 0.14 nM, 1 nM, 1.2 nM, 11 nM, or 100 nM.

[0134] Different assays for measuring CD40 activation are available, including the use of commercially available kits. In one example, the target cells are CHO cells, and the activation is measured using an NFκB reporter gene assay. In another example, the target cells are dendritic cells (DCs), and the CD40 activation is measured using IL-12 secretion, CD80, and CD86 expression. In yet another example, the target cells are B cells, and the CD40 activation is measured using Ki67 and / or CD86 expression.

[0135] Also provided are exemplary 5T4-dependent CD40 agonist antibodies that are as long as proteins (e.g., multispecific antibodies, chimeric antigen receptors (CAR)). In one embodiment of the present disclosure, single-domain antibodies and polypeptides comprising such single-domain antibodies are provided. In one embodiment of the present disclosure, single-domain antibodies or polypeptides comprising the single-domain antibodies are provided, wherein the single-domain antibodies comprise CDR1, CDR2, and CDR3, each having the CDR1, CDR2, and CDR3 sequences of antibody 42p155 (SEQ ID NO: 1). In some embodiments, the CDR1, CDR2, and CDR3 comprise the amino acid sequences of SEQ ID NOs: 14-16, respectively.

[0136] Analysis has shown that certain residues in the CDR2 of 42p155 may undergo post-translational modification (PTM). Therefore, mutations are made to prevent such PTMs (hence the term PTM de-risking form), including NG=>NA or QG. See, for example, SEQ ID NOs: 63 and 64. Thus, in some embodiments, the CDR1, CDR2, and CDR3 comprise the amino acid sequences of SEQ ID NOs: 14, 63, and 16, respectively. Thus, in some embodiments, the CDR1, CDR2, and CDR3 comprise the amino acid sequences of SEQ ID NOs: 14, 64, and 16, respectively.

[0137] In some embodiments, humanized forms of 42p155 and their PTM-derisked counterparts are also provided, such as those provided in SEQ ID NOs: 53-62. In some embodiments, the humanized antibodies include a back mutation selected from 1P, 2S, 88P, and 98Q according to Kabat numbering. In some embodiments, the humanized antibodies include a back mutation 98Q. In some embodiments, the humanized antibodies include back mutations 88P and 98Q. In some embodiments, the humanized antibodies include back mutations 1P, 2S, 88P, and 98Q.

[0138] In some embodiments, in a humanized form, CDR1 comprises the amino acid sequence of SEQ ID NO: 14, CDR2 comprises the amino acid sequence of SEQ ID NO: 15, and CDR3 comprises the amino acid sequence of SEQ ID NO: 16. In some embodiments, the antibody or polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 53, 54, 57, and 60. In some embodiments, the antibody comprises the recited CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 53, 54, 57, and 60.

[0139] In some embodiments, in a humanized form, CDR1 comprises the amino acid sequence of SEQ ID NO: 14, CDR2 comprises the amino acid sequence of SEQ ID NO: 63, and CDR3 comprises the amino acid sequence of SEQ ID NO: 16. In some embodiments, the antibody or polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 55, 58, and 61. In some embodiments, the antibody comprises the recited CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 55, 58, and 61.

[0140] In some embodiments, in a humanized form, CDR1 comprises the amino acid sequence of SEQ ID NO: 14, CDR2 comprises the amino acid sequence of SEQ ID NO: 64, and CDR3 comprises the amino acid sequence of SEQ ID NO: 16. In some embodiments, the antibody or polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 56, 59, and 62. In some embodiments, the antibody comprises the recited CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 56, 59, and 62.

[0141] In some embodiments, anti-CD40 antibodies and antigen-binding fragments are also provided that compete with any of the antibodies of the present disclosure for binding to human CD40. In some embodiments, anti-CD40 antibodies and antigen-binding fragments are also provided that bind to the same epitope as any of the antibodies of the present disclosure. In some embodiments, anti-CD40 antibodies and antigen-binding fragments are also provided that comprise the CDR1, CDR2, and CDR3 of an antibody of the present disclosure.

[0142] It will also be understood by those of ordinary skill in the art that the antibodies disclosed herein can be modified so that they differ in amino acid sequence from the naturally occurring binding polypeptides from which they are derived. For example, a polypeptide or amino acid sequence derived from a specified protein can be similar, for example, having a certain percentage of identity with the starting sequence, for example, it can be 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identical to the starting sequence. In some embodiments, the modified antibodies or fragments retain the specified CDR sequences.

[0143] B. Bispecific Antibodies with Anti-5T4 Portions Targeting Segment (Bin) A or B

[0144] According to one embodiment of the present disclosure, a bispecific antibody or a multispecific antibody incorporating the bispecific antibody is provided, which includes an anti-5T4 portion and an anti-CD40 portion. In some embodiments, the anti-5T4 portion includes one or more anti-5T4 antibodies or fragments that compete with segment A or segment B antibodies for binding to human 5T4 protein.

[0145] As provided in Example 10, all anti-5T4 antibodies disclosed herein can be classified into four segments, A to D, based on binding competition assays. Segment A includes antibodies from naptumomab, as well as the new antibodies 14G12 and 393E9; segment B includes 159D5, and segment D includes 286B4. As reported in Example 12, antibodies in segments B and C exhibited excellent agonist activity.

[0146] Anti-5T4 antibodies and antigen-binding fragments of segment A can be denoted as antibody 14G12, humanized and de-risked versions thereof, and those that compete with 14G12 for binding to human 5T4 protein.

[0147] In one embodiment of the present disclosure, the anti-5T4 protein comprises an antibody or antigen-binding fragment comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region comprises CDR1, CDR2 and CDR3, and the light chain variable region comprises CDR1, CDR2 and CDR3. In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VLCDR3 respectively comprise the sequences of SEQ ID NOs: 75-80.

[0148] In some embodiments, the VH and VL comprise the sequences of SEQ ID NOs: 73 and 74, respectively. In some embodiments, the VH comprises the sequence of any one of SEQ ID NOs: 81-90, and the VL comprises the sequence of any one of SEQ ID NOs: 91-100. In some embodiments, the VH comprises the listed VH CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 73 and 81-90, and the VL comprises the listed VL CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 74 and 91-100.

[0149] In some embodiments, the VH and VL comprise the sequences of SEQ ID NOs: 83 and 91, respectively. In some embodiments, the VH comprises the listed VH CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 83, and the VL comprises the listed VL CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 91.

[0150] In some embodiments, the VH and VL comprise the sequences of SEQ ID NOs: 89 and 91, respectively. In some embodiments, the VH comprises the listed VH CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 89, and the VL comprises the listed VL CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 91.

[0151] Anti-5T4 antibodies and antigen-binding fragments of segment A may also be denoted as antibody 393E9, humanized and de-risked versions thereof, and those that compete with 393E9 for binding to human 5T4 protein.

[0152] In one embodiment of the present disclosure, the anti-5T4 protein comprises an antibody or antigen-binding fragment comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region comprises CDR1, CDR2 and CDR3, and the light chain variable region comprises CDR1, CDR2 and CDR3. In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VLCDR3 respectively comprise the sequences of SEQ ID NOs: 103-108.

[0153] In some embodiments, the VH and VL comprise the sequences of SEQ ID NOs: 101 and 102, respectively. In some embodiments, the VH comprises the sequence of any one of SEQ ID NOs: 109-115, and the VL comprises the sequence of any one of SEQ ID NOs: 116-120. In some embodiments, the VH comprises the listed VH CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 101 and 109-115, and the VL comprises the listed VL CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 102 and 116-120.

[0154] In some embodiments, the VH and VL comprise the sequences of SEQ ID NOs: 113 and 120, respectively. In some embodiments, the VH comprises the listed VH CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 113, and the VL comprises the listed VL CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 120.

[0155] Anti-5T4 antibodies and antigen-binding fragments of segment B can be denoted as antibody 159D5, humanized and de-risked versions thereof, and those that compete with 159D5 for binding to human 5T4 protein.

[0156] In one embodiment of the present disclosure, the anti-5T4 protein comprises an antibody or antigen-binding fragment comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region comprises CDR1, CDR2 and CDR3, and the light chain variable region comprises CDR1, CDR2 and CDR3. In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VLCDR3 respectively comprise the sequences of SEQ ID NOs: 123-128.

[0157] In some embodiments, the VH and VL comprise the sequences of SEQ ID NOs: 121 and 122, respectively. In some embodiments, the VH comprises the sequence of any one of SEQ ID NOs: 129-131, and the VL comprises the sequence of any one of SEQ ID NOs: 132-137. In some embodiments, the VH comprises the listed VH CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 121 and 129-131, and the VL comprises the listed VL CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 122 and 132-137.

[0158] In some embodiments, the VH and VL comprise the sequences of SEQ ID NOs: 130 and 133, respectively. In some embodiments, the VH comprises the listed VH CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 130, and the VL comprises the listed VL CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 133.

[0159] In certain embodiments, for certain desired activities, the anti-5T4 portion may include an antibody or antigen-binding fragment of segment D, which may be represented by antibody 286B4, humanized and de-risked forms thereof, and those that compete with 286B4 for binding to human 5T4 protein.

[0160] In one embodiment of the present disclosure, the anti-5T4 protein comprises an antibody or antigen-binding fragment comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region comprises CDR1, CDR2 and CDR3, and the light chain variable region comprises CDR1, CDR2 and CDR3. In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VLCDR3 respectively comprise the sequences of SEQ ID NOs: 140-145.

[0161] In some embodiments, the VH and VL comprise the sequences of SEQ ID NOs: 138 and 139, respectively. In some embodiments, the VH comprises the sequence of any one of SEQ ID NOs: 146-151, and the VL comprises the sequence of any one of SEQ ID NOs: 152-157. In some embodiments, the VH comprises the listed VH CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 138 and 146-151, and the VL comprises the listed VL CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 139 and 152-157. C. Bispecific Antibodies of Formats b11, b16, and b18

[0162] Various bispecific antibody formats have been tested in the accompanying examples. Format b11 ( Figure 4 and Figure 22 A) showed excellent activity characteristics, while format b16 ( Figure 22 D) and b18( Figure 22 F) shows even better performance.

[0163] In format b11, each of the two anti-CD40 VHH antibodies is fused to the C-terminus of a conventional anti-5T4 antibody. This 2+2 format is thus bivalent for both 5T4 and CD40. The Fc portion of the conventional anti-5T4 antibody can optionally be mutated to abrogate ADCC / CDC activity or to prevent binding to FcγRs. Exemplary mutations include leucine (L) to alanine (A) substitutions at positions 234 and 235 (LALA) and an alanine (A) to asparagine (N) substitution at position 297 (N297A).

[0164] In format b16, one of the VH / VL pairs in the conventional Fab 5T4 antibody is replaced by two anti-CD40 VHH antibodies in series. This 1+2 format is therefore a heterodimer. The Fc portion can optionally be mutated to lose ADCC / CDC activity or to prevent binding to FcγRs. Exemplary mutations include leucine (L) to alanine (A) substitutions (LALA) at positions 234 and 235 and an alanine (A) to asparagine (N) substitution at position 297 (N297A). In addition, the Fc fragment can be mutated to incorporate a knob-in-hole to reduce mispairing.

[0165] Format b18 differs from format b16 in that the anti-CD40 arm contains three, rather than just two, VHH anti-CD40 antibodies. Similarly, the Fc portion can optionally be mutated to abrogate ADCC / CDC activity or prevent binding to FcγRs. Exemplary mutations include leucine (L) to alanine (A) substitutions (LALA) at positions 234 and 235 and an alanine (A) to asparagine (N) substitution at position 297 (N297A). Furthermore, the Fc fragment can be mutated to incorporate a knob-in-hole to reduce mispairing.

[0166] According to one embodiment of the present disclosure, a bispecific antibody or a multispecific antibody incorporating the bispecific antibody is provided, comprising an anti-5T4 portion and an anti-CD40 portion. In some embodiments, the anti-5T4 portion comprises a conventional heavy chain-light chain pair. In some embodiments, the anti-CD40 portion comprises at least two consecutively fused VHH antibodies. In some embodiments, the anti-CD40 portion comprises at least three consecutively fused VHH antibodies. In some embodiments, a peptide chain comprising at least two or three VHH antibodies is fused to the N-terminus of one of the two chains of the Fc fragment.

[0167] In some embodiments, the bispecific antibody has a single 5T4 binding site. In some embodiments, the bispecific antibody has two, or three or more CD40 binding sites. In some embodiments, the Fc fragment is mutated to lose ADCC / CDC activity or prevent it from binding to FcγR. Exemplary mutations include leucine (L) to alanine (A) substitutions (LALA) at positions 234 and 235 and alanine (A) to asparagine (N) substitutions (N297A) at position 297. In addition, the Fc fragment can be mutated to incorporate a knob-in-hole to reduce mispairing.

[0168] In some embodiments, a bispecific antibody or a multispecific antibody incorporating the bispecific antibody is provided, comprising an anti-5T4 portion and an anti-CD40 portion. In some embodiments, the anti-5T4 portion comprises a conventional Fab antibody. In some embodiments, the anti-CD40 portion comprises two separate VHH antibodies, each of which is fused to the C-terminus of one of the two chains of an Fc fragment.

[0169] In some embodiments, the bispecific antibody has two 5T4 binding sites and two CD40 binding sites. In some embodiments, the Fc fragment is mutated to lose ADCC / CDC activity or prevent it from binding to FcγR. Exemplary mutations include leucine (L) to alanine (A) substitutions (LALA) at positions 234 and 235 and alanine (A) to asparagine (N) substitutions (N297A) at position 297. In addition, the Fc fragment can be mutated to incorporate a knob-in-hole to reduce mispairing.

[0170] Exemplary sequences of anti-5T4 antibodies and fragments, as well as exemplary sequences of anti-CD40 VHH antibodies, are provided throughout the disclosure and incorporated herein.

[0171] Chimeric Antigen Receptor (CAR)

[0172] Also provided is a chimeric antigen receptor (CAR) comprising a nanobody of the present disclosure. In CAR, nanobodies can serve as antigen recognition domains. In addition, in some embodiments, CAR further comprises an extracellular hinge region, a transmembrane domain, and an intracellular T cell signaling domain.

[0173] Hinge, also known as spacer, is a small domain located between the antigen recognition region and the cell outer membrane. Suitable hinges enhance the flexibility of the scFv receptor head and reduce the spatial constraints between the CAR and its target antigen. Exemplary hinge sequences are based on membrane-proximal regions from immune molecules such as IgG, CD8, and CD28.

[0174] The transmembrane domain is a structural component composed of hydrophobic α helices that span the cell membrane. It anchors the CAR to the plasma membrane, bridging the extracellular hinge and antigen recognition domain with the intracellular signaling region. Typically, a transmembrane domain from the membrane-proximal component of the intracellular domain can be used, such as the CD28 transmembrane domain.

[0175] The intracellular T cell signaling domain is located in the intracellular domain of the receptor, that is, inside the cell. After the antigen binds to the external antigen recognition domain, the CAR receptors cluster together and transmit the activation signal. The internal cytoplasmic end of the receptor then maintains signal transduction within the T cell. To mimic this process, the CD3-ζ cytoplasmic domain is often used as the main CAR intracellular domain component.

[0176] In addition to CD3 signaling, T cells also require co-stimulatory molecules in order to persist after activation. In some embodiments, the intracellular domain of the CAR receptor also includes one or more chimeric domains from co-stimulatory proteins such as CD28, CD27, CD134 (OX40) and CD137 (4-1BB).

[0177] Polynucleotides encoding antibodies and methods for preparing antibodies

[0178] The present disclosure also provides isolated polynucleotides or nucleic acid molecules encoding antibodies, variants or derivatives thereof of the present disclosure. The polynucleotides of the present disclosure can encode the entire heavy chain and light chain variable regions of an antigen-binding polypeptide, variants or derivatives thereof on the same polynucleotide molecule or on separate polynucleotide molecules. In addition, the polynucleotides of the present disclosure can encode portions of the heavy chain and light chain variable regions of an antigen-binding polypeptide, variants or derivatives thereof on the same polynucleotide molecule or on separate polynucleotide molecules.

[0179] In some embodiments, the polynucleotide is an mRNA molecule. In some embodiments, the mRNA can be introduced into a target cell to express the antibody or fragment thereof.

[0180] mRNA can be synthesized according to any of a variety of known methods. For example, mRNA can be synthesized by in vitro transcription (IVT). Briefly, IVT is typically performed using a linear or circular DNA template containing a promoter, a ribonucleoside triphosphate pool, a buffer system that may include DTT and magnesium ions, and an appropriate RNA polymerase (e.g., T3, T7, or SP6 RNA polymerase), DNase I, pyrophosphatase, and / or an RNase inhibitor. The exact conditions will vary depending on the specific application.

[0181] In some embodiments, to prepare mRNA encoding an antibody, the DNA template is transcribed in vitro. Suitable DNA templates typically have a promoter for in vitro transcription, such as a T3, T7, or SP6 promoter, followed by a desired nucleotide sequence and a termination signal for encoding the mRNA of the desired antibody (e.g., encoding a heavy chain or a light chain).

[0182] Can measure the mRNA sequence of desired antibody (for example, encoding heavy chain or light chain), and use standard method to mix it in DNA template.For example, starting from desired amino acid sequence (for example, desired heavy chain or light chain sequence), carry out virtual reverse translation based on degenerate genetic code.Then can use optimization algorithm to select suitable codon.Usually, can optimize G / C content to realize the highest possible G / C content on the one hand, consider the frequency of tRNA as far as possible according to codon use on the other hand.The RNA sequence of optimization, for example, can be set up and demonstrated by suitable display equipment, and compared with original (wild type) sequence.Also can analyze secondary structure to calculate respectively the stable and destabilizing characteristic or zone of RNA.

[0183] mRNA can be synthesized as unmodified or modified mRNA. Typically, mRNA is modified to enhance stability. Modifications of mRNA may include, for example, modifications of RNA nucleotides. Modified mRNA may therefore include, for example, backbone modifications, sugar modifications, or base modifications. In some embodiments, mRNA encoding an antibody (e.g., mRNA encoding heavy and light chains) may be synthesized from naturally occurring nucleotides and / or nucleotide analogs (modified nucleotides), including but not limited to purines (adenine (A), guanine (G)) or pyrimidines (thymine (T), cytosine (C), uracil (U)), as well as modified nucleotide analogs or derivatives of purines and pyrimidines, such as, for example, 1-methyl-adenine, 2-Methyl-adenine, 2-methylthio-N-6-isopentenyl-adenine, N6-methyl-adenine, N6-isopentenyl-adenine, 2-thio-cytosine, 3-methyl-cytosine, 4-acetyl-cytosine, 5-methyl-cytosine, 2,6-diaminopurine, 1-methyl-guanine, 2-methyl-guanine, 2,2-dimethyl-guanine, 7-methyl-guanine, inosine, 1-methyl-inosine, pseudouracil (5- uracil), dihydro-uracil, 2-thio-uracil, 4-thio-uracil, 5-carboxymethylaminomethyl-2-thio-uracil, 5-(carboxyhydroxymethyl)-uracil, 5-fluoro-uracil, 5-bromo-uracil, 5-carboxymethylaminomethyl-uracil, 5-methyl-2-thio-uracil, 5-methyl-uracil, N-uracil-5-oxyacetate, 5-methylaminomethyl-uracil, 5-methoxyaminomethyl-2 -thio-uracil, 5'-methoxycarbonylmethyl-uracil, 5-methoxy-uracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 1-methyl-pseudouracil, queosine, 13-D-mannosyl-queosine, wybutoxosine and phosphoramidates, phosphorothioates, peptide nucleotides, methylphosphonates, 7-deazaguanosine, 5-methylcytosine and inosine. The preparation of such analogs is known to those skilled in the art, for example, from U.S. Patent Nos. 4,373,071, 4,401,796, 4,415,732, 4,458,066, 4,500,707, 4,668,777, 4,973,679, 5,047,524, 5,132,418, 5,153,319, 5,262,530, and 5,700,642, the disclosures of which are incorporated by reference in their entirety.

[0184] In some embodiments, mRNA (for example, mRNA encoding heavy and light chains) can contain RNA backbone modifications. Generally, backbone modifications are chemically modified modifications of the phosphoric acid of the nucleotide backbone contained in the RNA. Exemplary backbone modifications generally include but are not limited to modifications from methylphosphonate, methylphosphoramidate, phosphoramidate, thiophosphate (for example, cytidine 5'-O-(1-thiophosphate)), borophosphate, positively charged guanidine radicals, etc., which means replacing phosphodiester bonds with other anions, cations, or neutral groups.

[0185] In some embodiments, mRNA (e.g., mRNA encoding heavy and light chains) may contain sugar modifications. Typical sugar modifications are chemical modifications of the sugars of the nucleotides, including but not limited to 2'-deoxy-2'-fluoro-oligoribonucleotides (2'-fluoro-2'-deoxycytidine 5'-triphosphate, 2'-fluoro-2'-deoxyuridine 5'-triphosphate), 2'-deoxy-2'-deamine-oligoribonucleotides (2'-amino-2'-deoxycytidine 5'-triphosphate, 2'-amino-2'-deoxyuridine 5'-triphosphate), 2'-O-alkyl oligoribonucleotides. Sugar modifications of nucleotides, 2'-deoxy-2'-C-alkyl oligoribonucleotides (2'-O-methylcytidine 5'-triphosphate, 2'-methyluridine 5'-triphosphate), 2'-C-alkyl oligoribonucleotides and their isomers (2'-aromatic cytidine 5'-triphosphate, 2'-aromatic uridine 5'-triphosphate) or azido triphosphate (2'-azido-2'-deoxycytidine 5'-triphosphate, 2'-azido-2'-deoxyuridine 5'-triphosphate).

[0186] In some embodiments, mRNA (for example, mRNA encoding heavy and light chains) may contain modifications of the bases of nucleotides (base modifications). Modified nucleotides containing base modifications are also referred to as base-modified nucleotides. Such base-modified nucleotides include but are not limited to 2-amino-6-chloropurine nucleoside 5'-triphosphate, 2-aminoadenosine 5'-triphosphate, 2-thiocytidine 5'-triphosphate, 2-thiouridine 5'-triphosphate, 4-thiouridine 5'-triphosphate, 5-aminoallylcytidine 5'-triphosphate, 5-aminoallyluridine 5'-triphosphate, 5-bromocytidine 5'-triphosphate, 5-bromouridine 5'-triphosphate, 5-iodine 5'-triphosphate, 5-iodouridine 5'-triphosphate, 5-methylcytidine 5'-triphosphate, 5-methyluridine 5'-triphosphate , 6-azacytidine 5'-triphosphate, 6-azauridine 5'-triphosphate, 6-chloropurine nucleoside 5'-triphosphate, 7-deazaadenosine 5'-triphosphate, 7-deazaguanosine 5'-triphosphate, 8-azaadenosine 5'-triphosphate, 8-azidoadenosine 5'-triphosphate, benzimidazole nucleoside 5'-triphosphate, N1-methyladenosine 5'-triphosphate, N1-methylguanosine 5'-triphosphate, N6-methyladenosine 5'-triphosphate, O6-methylguanosine 5'-triphosphate, pseudouridine 5'-triphosphate, puromycin 5'-triphosphate or xanthosine 5'-triphosphate.

[0187] Typically, mRNA synthesis involves the addition of a "cap" at the N-terminus (5') and a "tail" at the C-terminus (3'). The presence of the cap is crucial for providing resistance to nucleases found in most eukaryotic cells. The presence of the "tail" serves to protect the mRNA from degradation by exonucleases.

[0188] Thus, in some embodiments, an mRNA (e.g., an mRNA encoding a heavy chain and a light chain) comprises a 5' cap structure. The 5' cap is typically added as follows: first, an RNA terminal phosphatase removes one terminal phosphate group from the 5' nucleotide, leaving two terminal phosphates; then guanosine triphosphate (GTP) is added to the terminal phosphate via a guanylyltransferase, resulting in a 5'5'5 triphosphate bond; and then the 7-nitrogen of guanine is methylated by a methyltransferase. Examples of cap structures include, but are not limited to, m7G(5')ppp(5'(A, G(5')ppp(5)A and G(5)ppp(5')G.

[0189] In some embodiments, mRNA (e.g., mRNA encoding heavy and light chains) comprises a 3' poly (A) tail structure. The poly A tail on the 3' end of the mRNA generally comprises about 10 to 300 adenosine nucleotides (e.g., about 10 to 200 adenosine nucleotides, about 10 to 175 adenosine nucleotides, about 10 to 150 adenosine nucleotides, about 10 to 125 adenosine nucleotides, 10 to 100 adenosine nucleotides, about 10 to 75 adenosine nucleotides, about 20 to 70 adenosine nucleotides, or about 20 to 60 adenosine nucleotides). In some embodiments, mRNA encoding an antibody (e.g., mRNA encoding heavy and light chains) comprises a 3' poly (C) tail structure. Suitable poly-C tails on the 3' end of an mRNA typically contain about 10 to 200 cytosine nucleotides (e.g., about 10 to 150 cytosine nucleotides, about 10 to 100 cytosine nucleotides, about 20 to 70 cytosine nucleotides, about 20 to 60 cytosine nucleotides, or about 10 to 40 cytosine nucleotides). The poly-C tail can be added to the poly-A tail or can replace the poly-A tail.

[0190] In some embodiments, mRNA (for example, mRNA encoding heavy chain and light chain) includes 5' and / or 3' untranslated region.In some embodiments, 5' untranslated region includes one or more elements affecting mRNA stability or translation, such as iron response element.In some embodiments, 5' untranslated region can be a length of about 50 to 500 nucleotides (for example, a length of about 50 to 400 nucleotides, a length of about 50 to 300 nucleotides, a length of about 50 to 200 nucleotides or a length of about 50 to 100 nucleotides).

[0191] In some embodiments, the 5' region of the mRNA (e.g., mRNA encoding the heavy and light chains) comprises a sequence encoding a signal peptide, such as those described herein. In a specific embodiment, a signal peptide derived from human growth hormone (hGH) is incorporated into the 5' region. Typically, the signal peptide coding sequence is linked directly or indirectly to the heavy or light chain coding sequence at the N-terminus.

[0192] The present technology can be used to deliver any antibody known in the art and antibodies to desired antigens that can be generated using standard methods. The present invention can be used to deliver monoclonal antibodies, polyclonal antibodies, antibody mixtures or cocktails, human antibodies or humanized antibodies, chimeric antibodies or bispecific antibodies.

[0193] Methods for preparing antibodies are well known in the art and are described herein. In certain embodiments, the variable and constant regions of the antigen-binding polypeptides of the present disclosure are fully human. Fully human antibodies can be prepared using techniques described in the art and as described herein. For example, fully human antibodies against specific antigens can be prepared by administering an antigen to a transgenic animal that has been modified to produce such antibodies in response to an antigen attack, but whose endogenous loci have been disabled. Exemplary techniques for preparing such antibodies are described in U.S. Patents 6,150,584, 6,458,592, and 6,420,140, ​​which are incorporated by reference in their entirety.

[0194] In certain embodiments, the prepared antibodies do not induce a harmful immune response in the animal to be treated (e.g., in humans). In one embodiment, the antigen-binding polypeptides, variants, or derivatives thereof disclosed herein are modified using techniques recognized in the art to reduce their immunogenicity. For example, antibodies can be humanized, primatized, deimmunized, or chimeric antibodies can be prepared. These types of antibodies are derived from non-human antibodies, typically murine or primate antibodies, which retain or substantially retain the antigen-binding properties of the parent antibody, but have lower immunogenicity in humans. This can be achieved by various methods, including (a) transplanting the entire non-human variable domain onto a human constant region to produce a chimeric antibody; (b) transplanting at least a portion of one or more non-human complementary determining regions (CDRs) into a human framework region and constant region with or without retaining key framework residues; or (c) transplanting the entire non-human variable domain, but "masking" them with human-like segments by replacing surface residues. Such methods are disclosed in Morrison et al., Proc. Natl. Acad. Sci. USA 57:6851-6855 (1984); Morrison et al., Adv. Immunol. 44:65-92 (1988); Verhoeyen et al., Science 239:1534-1536 (1988); Padlan, Molec. Immun. 25:489-498 (1991); Padlan, Molec. Immun. 31:169-217 (1994) and U.S. Pat. Nos. 5,585,089, 5,693,761, 5,693,762 and 6,190,370, all of which are hereby incorporated by reference in their entirety.

[0195] Deimmunization can also be used to reduce the immunogenicity of antibodies. As used herein, the term "deimmunization" includes changing antibodies to modify T cell epitopes (see, for example, International Application Publication Nos. WO / 9852976A1 and WO / 0034317A2). For example, the variable heavy chain and variable light chain sequences from the starting antibody are analyzed, and human T cell epitope "atlas" is produced from each V district, which shows the position of the epitope related to the complementary determining region (CDR) and other key residues in the sequence. Single T cell epitopes from the T cell epitope atlas are analyzed to identify alternative amino acid substitutions with a low risk of changing the activity of the final antibody. A series of alternative variable heavy chain sequences and variable light chain sequences have been designed, and the design includes combining amino acid substitutions, and subsequently these sequences are incorporated into a series of binding polypeptides. Typically, 12 to 24 variant antibodies are produced and the combination and / or function of these antibodies are tested. The complete heavy chain and light chain genes comprising modified variable regions and human constant regions are then cloned into expression vectors, and subsequently plasmids are introduced into cell lines to produce complete antibodies. The antibodies are then compared in appropriate biochemical and biological assays, and the optimal variant is identified.

[0196] The binding specificity of the antigen-binding polypeptides of the present disclosure can be determined by in vitro assays, such as immunoprecipitation, radioimmunoassay (RIA), or enzyme-linked immunosorbent assay (ELISA).

[0197] Treatment of tumors, especially cold tumors

[0198] As described herein, the antibodies, variants, or derivatives of the present disclosure may be used in certain therapeutic and diagnostic methods.

[0199] The present disclosure also relates to antibody-based therapies, which involve administering the antibodies of the present disclosure to patients, such as animals, mammals, and humans, for treating one or more disorders or conditions described herein. The therapeutic compounds of the present disclosure include, but are not limited to, antibodies of the present disclosure (including variants and derivatives thereof as described herein) and nucleic acids or polynucleotides encoding antibodies of the present disclosure (including variants and derivatives thereof as described herein).

[0200] The antibodies disclosed herein can also be used to treat or inhibit cancer. In some embodiments, tumor antigens (e.g., tight junction protein 18.2) are overexpressed in tumor cells. Therefore, in some embodiments, a method for treating cancer in a patient in need is provided. The method, in one embodiment, requires administering an effective amount of an antibody disclosed herein to the patient. In some embodiments, at least one of the patient's cancer cells (e.g., stromal cells) expresses, overexpresses, or is induced to express a tumor antigen. Induced gene expression can be performed, for example, by administering a tumor vaccine or radiotherapy.

[0201] Tumors that can be appropriately treated include those in bladder cancer, non-small cell lung cancer, kidney cancer, breast cancer, urethral cancer, colorectal cancer, head and neck cancer, squamous cell carcinoma, Merkel cell carcinoma, gastrointestinal cancer, gastric cancer, esophageal cancer, ovarian cancer, renal cancer and small cell lung cancer. Therefore, the antibodies of the present disclosure can be used to treat any one or more of these cancers.

[0202] In some embodiments, the tumors treated are those that are particularly challenging to treat with conventional immuno-oncology therapies, such as with antibodies targeting immune checkpoints (ICPs). Sometimes, such tumors are referred to as "cold tumors" or "non-immunogenic tumors." CD40 activation can convert so-called cold tumors (non-immunogenic tumors) into hot tumors. In some embodiments, the present disclosure therefore provides methods and uses for treating cold tumors with the antibodies disclosed herein.

[0203] In some embodiments, non-immunogenic tumors are tumors that are not infiltrated by T cells, or are tumors that are defective in T cell filtration, antigen presenting cells (APCs), or T cell activation, or are tumors that are defective in T cell homing to the tumor bed. All prostate cancers, pancreatic cancers, and leukemias are non-immunogenic. The vast majority of breast cancers (95%), colorectal cancers (95%), gastric cancers (87%), head and neck cancers (84%), liver cancers (83%), esophageal cancers (86%), cervical cancers (87%), and thyroid cancers (87%) are also non-immunogenic. In addition, 83% of lung cancers, 79% of bladder cancers, 77% of kidney cancers, 70% of uterine cancers, and 66% of melanomas are also non-immunogenic.

[0204] Identification of non-immunogenic or cold tumors can also be performed by measuring the type, density, and location of immune cells within the tumor. For example, Galon and Bruni (Nature Reviews Drug Discovery, Vol. 18, pp. 197–218 (2019)) described a standardized scoring system, Immunoscore, which guides the distinction between hot and cold tumors based on the quantification of two lymphocyte populations (CD40 and CD8) (e.g., in resected tissue). Immunoscore ranges from Immunoscore 0 (I0, low density, such as the lack of two cell types in two areas) to I4 (high immune cell density in two locations). By classifying cancers according to their immune infiltration, the scoring system provides an immune-based tumor classification, including the definition of "hot" (highly infiltrated, Immunoscore I4) tumors and "cold" (non-infiltrated, Immunoscore I0) tumors.

[0205] In some embodiments, the tumor is resistant to treatment with an immune checkpoint inhibitor (such as a PD-L1 inhibitor, a PD-1 inhibitor, a CTLA-4 inhibitor, or a combination thereof). In some embodiments, the cancer is prostate cancer, pancreatic cancer, or leukemia. In some embodiments, the cancer is breast cancer, colorectal cancer, gastric cancer, head and neck cancer, liver cancer, esophageal cancer, cervical cancer, or thyroid cancer. In some embodiments, the cancer is lung cancer, bladder cancer, kidney cancer, uterine cancer, or melanoma.

[0206] In some embodiments, patients treated with a CD40 agonist antibody (or multispecific antibody) of the present disclosure are further treated with a second anticancer agent. In some embodiments, the second anticancer agent is an immune checkpoint inhibitor, such as an antibody specific for PD-1, PD-L1, or CTLA-4, but not limited thereto. In some embodiments, the second anticancer agent is administered together with a CD40 agonist antibody (or multispecific antibody) of the present disclosure. In some embodiments, the second anticancer agent is administered before or after administration of a CD40 agonist antibody (or multispecific antibody) of the present disclosure.

[0207] Other disorders or conditions associated with increased cell survival that can be treated, prevented, diagnosed and / or prognosed using the antibodies of the present disclosure, or variants or derivatives thereof, include, but are not limited to, progression and / or metastasis of malignancies and related disorders such as leukemias (including acute leukemias (e.g., acute lymphocytic leukemia, acute myeloid leukemia (including myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, and erythroleukemia)) and chronic leukemias (e.g., chronic myeloid (granulocytic) leukemia). ) leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors, including but not limited to sarcomas and carcinomas, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, and sarcoma. tumor), leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, thyroid cancer, endometrial cancer, melanoma, prostate cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct cancer, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

[0208] The specific dosage and treatment regimen for any particular patient will depend on a variety of factors, including the specific antibody, variant or derivative used, the patient's age, weight, general health, sex and diet, as well as the time of administration, rate of excretion, drug combination and the severity of the specific disease being treated. The judgment of the medical professional on such factors is within the ordinary skill of the art. The amount will also depend on the individual patient to be treated, the route of administration, the type of formulation, the characteristics of the compound used, the severity of the disease and the desired effect. The amount used can be determined by pharmacological and pharmacokinetic principles well known in the art.

[0209] The method of administration of antibodies, variants may include but is not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural and oral routes. Antigen-binding polypeptides or compositions can be administered by any convenient route, such as by infusion or bolus injection, absorbed by epithelial or mucocutaneous linings (such as oral mucosa, rectum and intestinal mucosa, etc.), and can be administered together with other bioactive agents. Therefore, pharmaceutical compositions containing antigen-binding polypeptides of the present disclosure can be administered orally, rectally, parenterally, intracisternal, intravaginal, intraperitoneally, topically (such as by powder, ointment, drops or transdermal patch), buccally or as an oral or nasal spray.

[0210] The term "parenteral" as used herein refers to modes of administration that include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous, and intraarticular injection and infusion.

[0211] Administration may be systemic or local. In addition, it may be desirable to introduce the antibodies of the present disclosure into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter, for example, connected to a reservoir, such as an Ommaya reservoir. Pulmonary administration may also be employed, for example, by using an inhaler or nebulizer and a formulation containing an aerosolizing agent.

[0212] It may be desirable to administer the antibodies, polypeptides, or compositions of the present disclosure locally to the area in need of treatment; this can be achieved, for example, but not limited to, by local infusion during surgery, topical application (e.g., in conjunction with a wound dressing after surgery), by injection, by catheter, by suppository, or by implant, which is a porous, non-porous, or gel-like material, including membranes, such as sialic acid membranes or fibers. Preferably, when administering proteins (including antibodies) of the present disclosure, care must be taken to use materials that the proteins are not absorbed.

[0213] Composition

[0214] The present disclosure also provides pharmaceutical compositions. Such compositions comprise an effective amount of an antibody and an acceptable carrier.

[0215] In a specific embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. Additionally, a "pharmaceutically acceptable carrier" is generally any type of non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary.

[0216] The term "carrier" refers to a diluent, adjuvant, excipient or vehicle used together with the therapeutic agent. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including oils from petroleum, animal, plant or synthetic sources, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. When the pharmaceutical composition is administered intravenously, water is a preferred carrier. Saline solutions and aqueous glucose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skimmed milk powder, glycerol, propylene, ethylene glycol, water, ethanol, etc. If desired, the composition can also contain a small amount of wetting agent or emulsifier, or pH buffer, such as acetate, citrate or phosphate. Also contemplated are antimicrobial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; and tension modifiers such as sodium chloride or glucose. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, and the like. The compositions can be formulated as suppositories with conventional binders and carriers (such as triglycerides). Oral formulations can contain standard carriers such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences by EW Martin, which is incorporated herein by reference. Such compositions will contain a therapeutically effective amount of an antigen-binding polypeptide, preferably in purified form, and an appropriate amount of carrier to provide a form suitable for administration to a patient. The formulation should be suitable for the mode of administration. The parent formulation can be packaged in ampoules, disposable syringes, or multidose vials made of glass or plastic.

[0217] In one embodiment, according to conventional procedures, composition is formulated into a pharmaceutical composition suitable for intravenous administration to people. Usually, the composition for intravenous administration is a solution in a sterile isotonic aqueous buffer. If necessary, composition can also include a solubilizing agent and a local anesthetic (such as lidocaine) to alleviate the pain at the injection site. Usually, these compositions are provided separately or mixed together in unit dosage form, for example, in the form of a lyophilized powder or anhydrous concentrate in a sealed container (such as an ampoule or a pouch) indicating the amount of the active agent. In the case where the composition is used by infusion, it can be distributed with an infusion bottle containing sterile pharmaceutical grade water or saline. In the case where the composition is used by injection, the sterile water for injection or saline of an ampoule can be provided to mix each component before use.

[0218] The compounds of the present disclosure can be formulated in neutral or salt form. Pharmaceutically acceptable salts include those formed with anions, such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, and the like, and those formed with cations, such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, and the like.

[0219] Example

[0220] Example 1: Generation of VHH antibodies against human CD40

[0221] This example describes the generation of single domain (VHH) antibodies directed against the human CD40 protein.

[0222] Immunization: To generate VHH antibodies against human CD40, two alpacas were immunized with human CD40 protein. After four rounds of immunization, the antibody titers of the immunized alpaca sera were assessed by ELISA.

[0223] Immune library construction: Phage libraries were constructed using phagemid vectors consisting of VHH gene fragments amplified from PBMCs of CD40-immunized alpacas. The antibody format was VHH fragments in a phage display library. Four immune libraries were generated from PBMCs of different alpacas at different immunization rounds. The size of each library was greater than 1×10 8 , and sequence diversity analysis was performed as follows. 24 or 48 clones were selected from each library and further sequenced. The sequences showed that the CDRs of these four libraries had sufficient diversity.

[0224] Phage panning and clone selection: CD40 protein was used as the antigen for phage library panning.

[0225] Phage library solution panning against human CD40: Bound phage were eluted with Gly-HCl. The resulting phage was designated Output 1. The bound phage were incubated with SS320 cells and plated onto 2YT plates for the next round of panning. A total of three rounds of panning were performed. After three rounds of screening, phage ELISAs for Outputs 1, 2, and 3 showed enrichment of CD40 binders.

[0226] Individual clones were selected from output 2 and output 3 phage. Antigen binding ELISA was performed on these cloned phage. Clones showing good binding efficacy were selected for subsequent sequencing.

[0227] Thirteen candidate sequences were cloned into the pcDNA 3.4 vector and expressed in 293F cells. Monoclonal antibodies were purified from the culture supernatant by protein G. Purified antibodies were evaluated by ELISA binding to CD40-His protein.

[0228] The amino acid sequences of the single variable domains of 42p155, 2p834, 2p931, 42p655, 2p1294, 2p957, 42p495, 3p78, 2p415, 2p442, 2p551, 2p80, and 2pl 130 are listed in Table 1 and Tables 1A to 1E below.

[0229] Table 1. Variable domain sequences

[0230]

[0231]

[0232] Table 1A. CDR sequences of 42p155

[0233] name sequence SEQ ID NO: 42p155-CDR1 SYTMS 14 42p155-CDR2 TITHNGAITTYAESAQG 15 42p155-CDR3 GGGSNYYRES 16

[0234] Table 1B. CDR sequences of 2p834

[0235] name sequence SEQ ID NO: 2p834-CDR1 RYTMS 17 2p834-CDR2 TITHNGSITTYAESAQG 18 2p834-CDR3 GGGSNYYRES 19

[0236] Table 1C.2p931 CDR sequences

[0237] name sequence SEQ ID NO: 2p931-CDR1 NYMMN 20 2p931-CDR2 SITSAGDITSYTESVKG 21 2p931-CDR3 GGCGNYYRES 22

[0238] Table 1D. CDR sequences of 42p655

[0239] name sequence SEQ ID NO: 42p655-CDR1 RYTMS 23 42p655-CDR2 SITDNGSITTYAESAQG 24 42p655-CDR3 GGGSNYYRES 25

[0240] Table 1 CDR sequences of E.2p1294

[0241] name sequence SEQ ID NO: 2p1294-CDR1 RYTMS 26 2p1294-CDR2 AISDNGAITTYTESAQG 27 2p1294-CDR3 GGGSNYYRES 28

[0242] Table 1F.2p957 CDR sequences

[0243] name sequence SEQ ID NO: 2p957-CDR1 NYIMS 29 2p957-CDR2 SITNSGGITSYTESVKG 30 2p957-CDR3 GGSDNYYRGS 31

[0244] Table 1 CDR sequences of G.42p495

[0245] name sequence SEQ ID NO: 42p495-CDR1 NSAMS 32 42p495-CDR2 TIYSGKSNTDYADSVKG 33 42p495-CDR3 GAASDWYVPRDY 34

[0246] Table 1H. CDR sequences of 3p78

[0247] name sequence SEQ ID NO: 3p78-CDR1 NYAMS 35 3p78-CDR2 TITHNGAITTYAESAQG 36 3p78-CDR3 GGGSNYYRES 37

[0248] Table 1I.2p415 CDR sequences

[0249]

[0250]

[0251] Table 1 CDR sequences of J.2p442

[0252] name sequence SEQ ID NO: 2p442-CDR1 YFAIG 41 2p442-CDR2 CISGGGSTRYADSVKG 42 2p442-CDR3 ARLLSRNCVPRDSGS 43

[0253] Table 1 CDR sequences of K.2p551

[0254] name sequence SEQ ID NO: 2p551-CDR1 YYAIG 44 2p551-CDR2 CISGGGSTRYADSVKG 45 2p551-CDR3 ARLLSTNCVPRDSGS 46

[0255] Table 1 CDR sequences of L.2p80

[0256] name sequence SEQ ID NO: 2p80-CDR1 SYAMS 47 2p80-CDR2 TIGWIGENTYYADSVKG 48 2p80-CDR3 GLPANRYYDY 49

[0257] Table 1 CDR sequences of M.2p1130

[0258] name sequence SEQ ID NO: 2p1130-CDR1 DYGIG 50 2p1130-CDR2 CITPNGLMMNFANTVGSVAG 51 2p1130-CDR3 SRDDSCRGSLSDYDD 52

[0259] Example 2: Binding activity to CD40 antigen

[0260] This example tests the binding activity of antibodies to CD40 protein.

[0261] 2.1 ELISA binding to CD40

[0262] To evaluate the binding activity of clones 42p155, 2p834, 2p931, 42p655, 2p1294, 2p957, 42p495, 3p78, 2p415, 2p442, 2p551, 2p80, and 2p1130, chimeric monospecific antibodies (mAbs) from these clones, as well as selumetumab, a clinical benchmark anti-CD40 agonist antibody, were tested in ELISA.

[0263] Briefly, microtiter plates were coated with 1.0 μg / ml human CD40-His protein in PBS at 100 μl / well at 4°C overnight and then blocked with 150 μl / well of 1% BSA. Ten-fold dilutions of 42p155, 2p834, 2p931, 42p655, 2p1294, 2p957, 42p495, 3p78, 2p415, 2p442, 2p551, 2p80, and 2p1130 antibodies starting at 100 nM were added to each well and incubated for 1 hour at room temperature. The plates were washed with PBS / Tween and then incubated with anti-human IgG (H&L) (GOAT) antibody peroxidase conjugated at room temperature for 30 minutes. After washing, the plates were developed with TMB substrate and analyzed by spectrophotometer at OD450 nm. Figure 1 As shown in Figure A and Table 2, all of these clones bound to human CD40 with high activity and comparable potency to selumetumab.

[0264] The binding affinity of these clones to cynomolgus monkey CD40 was also tested. Microtiter plates were coated with 1.0 μg / ml cynomolgus monkey CD40-His protein in PBS at 100 μl / well at 4°C overnight and then blocked with 150 μl / well of 1% BSA. Ten-fold dilutions of 42p155, 2p834, 2p931, 42p655, 2p1294, 2p957, 42p495, 3p78, 2p415, 2p442, 2p551, 2p80 and 2p1130 antibodies starting at 100 nM were added to each well and incubated at room temperature for 1 hour. The plates were washed with PBS / Tween and then incubated with conjugated anti-human IgG (H&L) (GOAT) antibody peroxidase at room temperature for 30 minutes. After washing, the plates were developed with TMB substrate and analyzed by spectrophotometer at OD 450 nm. Figure 1 As shown in Figure 2 and Table 2, all of these clones bound to cynomolgus CD40 with high activity and showed good cross-reactivity between human and cynomolgus CD40 proteins.

[0265] Table 2. Cross-species activity of clones

[0266] <![CDATA[克隆 / EC 50 (nM)]]> human CD40 Cynomolgus monkey CD40 42p155 1.73 0.90 2p834 0.76 0.20 2p931 0.99 0.26 42p655 1.24 0.35 2p1294 1.16 0.32 2p957 1.80 1.08 42p495 1.52 1.00 3p78 2.95 2.84 2p415 1.27 0.23 2p442 1.95 0.30 2p551 1.40 0.13 2p80 3.08 8.29 2p1130 2.56 0.73

[0267] --: Not combined

[0268] 2.2 Binding to cell surface CD40

[0269] To evaluate their binding affinity for cell surface CD40, chimeric anti-CD40 monospecific antibodies were tested by FACS in Jurkat cell lines overexpressing CD40. A total of 1 × 10 5 Jurkat-CD40 cells were incubated with 10-fold serial dilutions of antibodies starting from 100 nM at 4°C for 30 minutes. After washing with FACS buffer, PE-conjugated anti-human IgG antibodies were added to each well and incubated at 4°C for 30 minutes. After washing, the MFI of PE was evaluated by MACSQuant analyzer16. Figure 2 As shown, the tested antibodies showed concentration-dependent binding ability to CD40.

[0270] 2.3 Comprehensive protein dynamics of CD40

[0271] The antibodies were tested for binding to the anti-claudin 18.2 moiety directed against recombinant CD40 protein (human CD40-his tag) in either monospecific antibody format (cAb) or bispecific format (BiAb, e.g., Figure 4Bispecific antibodies targeting each of the 42p155, 2p834, 2p931, 42p655, 2p1294, 2p957, 42p495, 3p78, 2p415, 2p442, 2p551, and 2p1130 clones, as well as the 2p80 monospecific antibody, were captured using a Protein A chip. Serial dilutions of human CD40-His-tagged protein were injected over the captured antibodies at a flow rate of 30 μl / min for 2 minutes. Antigen dissociation was allowed for 6 minutes. All experiments were performed on a Biacore T200. Data analysis was performed using Biacore T200 evaluation software. The results are shown in Table 4 below. All antibodies exhibited moderate binding and varying association / dissociation patterns.

[0272] Table 4: Overall kinetics measured by Biacore

[0273]

[0274] 2.4 Cross-reactivity between OX40 and 4-1BB

[0275] To evaluate the cross-reactivity of the chimeric anti-CD40 antibodies to other family members of the TNF receptor, ELISA binding to human 4-1BB and human OX40 was performed.

[0276] In brief, microtiter plates were coated with 1 μg / ml human 4-1BB protein or human OX40 protein in PBS at 100 μl / well at 4 ° C overnight and then blocked with 150 μl / well 1% BSA. Ten-fold dilutions of 42p155, 2p834, 2p931, 42p655, 2p1294, 2p957, 42p495, 3p78, 2p415, 2p442, 2p551, 2p80 and 2p1130 antibodies starting from 100 nM were added to each well and incubated at room temperature for 1 hour. The plates were washed with PBS / Tween and then incubated with conjugated anti-human IgG (H&L) (GOAT) antibody peroxidase at room temperature for 30 minutes. After washing, the plates were developed with TMB substrate and analyzed by spectrophotometer at OD 450 nm. Figure 3 As shown, all of these clones showed no cross-reactivity to human 4-1BB or human OX40.

[0277] Example 3: Binding activity of the Claudin 18.2 / CD40 bispecific antibody to human CD40 on human dendritic cells and B cells

[0278] In this example, bispecific antibodies (BiAbs) comprising anti-CD40 nanobodies and anti-claudin 18.2 (CLDN18.2) units were generated and tested. Two anti-CD40 fragments with 42p155, 2p834, 2p931, 42p655, 2p1294, 2p957, 42p495, 3p78, 2p415, 2p442, 2p551, 2p80, or 2p1130 in VHH format (at the C-terminus) and two anti-claudin 18.2 units in Fab format (at the N-terminus) were constructed into a 2+2 bispecific antibody format (see Figure 4 FACS was used to evaluate the binding activity of the anti-Claudin 18.2 / CD40 bispecific antibody to CD40 on human dendritic cells (DCs) and human B cells.

[0279] 3.1 Binding activity of bispecific antibodies to human CD40 on human dendritic cells

[0280] Human DCs were induced from human CD14+ cells for 6 to 7 days in the presence of IL-4 and GM-CSF, and the cells were isolated from human peripheral blood mononuclear cells (PBMCs) using CD14 microbeads according to the manufacturer's protocol. The induced human DCs were first blocked with FcR blocking reagent (MACS) at 4°C for 15 minutes, and then incubated with 10-fold serial dilutions of claudin 18.2 / CD40 bispecific antibody starting at 100 nM at 4°C for 30 minutes. PE goat anti-human IgG Fc secondary antibody (eBioscience TM , Invitrogen) was added to each well and incubated at 4°C for 30 minutes. The samples were washed with FACS buffer and then incubated with APC mouse anti-human CD11C (BD) at 4°C for 30 minutes. The mean fluorescence intensity (MFI) of PE gated on CD11C+ cells was evaluated by MACSQuant analyzer 16.

[0281] The results are shown in Figure 5 The anti-CLDN18.2 / CD40 bispecific antibodies tested showed concentration-dependent binding to human DCs. The binding activity of the bispecific antibodies was weaker than that of the reference monospecific antibody, selumetumab.

[0282] 3.2 Binding activity of bispecific antibodies to human CD40 on human B cells

[0283] Human B cells were isolated from human PBMC using a B cell isolation kit according to the manufacturer's protocol. Human B cells and DCs were first blocked with FcR blocking reagent (MACS) at 4°C for 15 minutes and then incubated with 10-fold serial dilutions of CLDN18.2 / CD40 bispecific antibody starting at 100 nM at 4°C for 30 minutes. PE goat anti-human IgG Fc secondary antibody (eBioscience TM , Invitrogen) was added to each well and incubated at 4°C for 30 minutes. The samples were washed with FACS buffer and then incubated with APC mouse anti-human CD19 (BD) at 4°C for 30 minutes. The mean fluorescence intensity (MFI) of PE gated on CD19+ cells was evaluated by MACSQuant analyzer 16.

[0284] The results are shown in Figure 6 The tested anti-CLDN18.2 / CD40 bispecific antibodies showed concentration-dependent binding to human B cells. The binding activity of the bispecific antibodies was much weaker than that of the reference monospecific antibody, selumetumab.

[0285] Example 4. Functional activity of CD40 nanobody

[0286] This example tests the functional activity of the antibodies and shows that, unlike selukumab, the VHH chimeric antibody activates CD40 signaling only at low levels.

[0287] Cell line-based functional characterization of CD40 monoclonal antibodies

[0288] To evaluate the ability of CD40 monoclonal antibodies to activate the CD40 signaling pathway, a commercial CD40 NF-κB luciferase reporter gene system was used. In this assay, H_CD40 (TNFRSF5) NFκB-reporter Jurkat (Genomeditech, cat# GM-C09520) was used as a reporter cell line. The H_CD40 (TNFRSF5) NFκB-reporter Jurkat cell line was genetically modified to stably express CD40 and luciferase downstream of the response element. Luciferase expression is induced when the antibody binds to the CD40 receptor. Briefly, cells were plated at a density of 2.5×10 4 Reporter cells were cultured in a white 96-well plate at 10 cells / well. Antibodies were serially diluted 10-fold and added to the white 96-well assay plate at final concentrations ranging from 0.001 nM to 100 nM. After a 5-hour incubation at 37°C, luminescence was obtained by adding luciferase substrate and measured using a microplate reader. Four-parameter logistic curve analysis was performed using GraphPad software.

[0289] like Figure 7As shown, the selumetumab monoclonal antibody activated CD40 signaling in a dose-dependent manner. 42p155, 2p834, 2p931, 42p655, 2p1294, 2p957, 42p495, 3p78, 2p415, 2p442, 2p551, 2p80, and 2p1130 antibodies induced CD40 signaling only at doses above 10 nM, and their maximum RLU values ​​were all less than half of the highest value of selumetumab in the same experimental setting.

[0290] Example 5. Functional activity of anti-claudin 18.2 / CD40 bispecific antibody

[0291] In this example, the CD40 NF-κB luciferase reporter system was used to test Figure 4 The activity of the anti-Claudin 18.2 / CD40 bispecific antibody produced in the indicated format and its activity in promoting human dendritic cell and B cell immune responses.

[0292] 5.1 Cell Line-Based Functional Characterization of the Claudin 18.2-CD40 Bispecific Antibody

[0293] To evaluate the ability of the anti-claudin 18.2 / CD40 bispecific antibody to activate the CD40 signaling pathway, a commercial CD40 NF-κB luciferase reporter system was used. In this assay, H_CD40 (TNFRSF5) NFκB-reporter Jurkat (Genomeditech, cat# GM-C09520) was used as effector cells, and CHO-K1 cells expressing or not expressing claudin 18.2 were used as target cells. Briefly, cells were plated at a density of 2.0 × 10 4 Effector cells / well and 2.0×10 4 Target cells (E / T ratio = 1:1) were co-cultured in a white 96-well plate. Antibodies were serially diluted 10-fold and added to a white 96-well assay plate at a final concentration range of 0.001 nM to 100 nM. After incubation at 37°C for 5 hours, luminescence was obtained by adding luciferase substrate and measured by a microplate reader. Four-parameter logistic curve analysis was performed using GraphPad software.

[0294] like Figure 8As shown, the monoclonal antibody selenulumab can dose-dependently enhance CD40 signaling in cells overexpressing CHO-K1 and CHO-claudin 18.2. The activities of the anti-claudin 18.2 / CD40 bispecific antibodies 42p155-BiAb, 2p834-BiAb, 2p931-BiAb, 42p655-BiAb, 2p1294-BiAb, 2p957-BiAb, 42p495-BiAb, 3p78-BiAb, 2p415-BiAb, 2p442-BiAb, 2p551-BiAb, 2p80-BiAb, and 2p1130-BiAb are individually or partially dependent on the expression of claudin 18.2 on cells, and they exhibit much stronger activation of CD40 signaling in the presence of claudin 18.2. In addition, the bispecific antibodies exhibited a variety of different CD40 activities. Based on EC 50 and the highest value, CD40 clones were divided into four categories (grade 1 to grade 4), as shown in Table 5.

[0295] Table 5. Potency categories

[0296]

[0297] 5.2 Bispecific Antibodies Promote the Immune Response of Human Dendritic Cells

[0298] To investigate the ability of the Claudin 18.2-CD40 bispecific antibody to stimulate human dendritic cell (DC) responses, IL-12 cytokine release and CD80 / CD86 expression by DCs were examined.

[0299] Human DCs were obtained according to the procedure described in Example 3.1. Human DCs were used as effector cells. CHO-K1 cells expressing claudin 18.2 were used as target cells. Human DCs (5×10 4 ) and CHO-K1-claudin 18.2 or parental CHO-K1 cells (1.5×10 4 The bispecific antibodies were serially diluted 10-fold and added to the culture medium at a final concentration starting at 100 nM. After 48 hours of incubation, IL-12 levels in the culture medium were measured using the IL-12 / p40 (human) LANCE Ultra TR-FRET Detection Kit (PerkinElmer). Data were analyzed using nonlinear regression, a 4-parameter logistic equation.

[0300] The activation of DCs leads to the upregulation of the co-stimulatory molecules CD80 / 86. Here, the CD80 / CD86 expression of DCs was detected by FACS according to the staining and analysis procedures. Briefly, stimulated DCs were harvested by pipetting and washed with FACS buffer. PE mouse anti-human CD80, BV421 mouse anti-human CD86, and APC mouse anti-human CD11C (BD) were added to each well and incubated at 4°C for 30 minutes. After washing, the MFI of PE and BV421 gated on CD11C+ cells was evaluated by MACSQuant analyzer 16.

[0301] like Figure 9 and Figure 10 As shown, selenulumab monoclonal antibody can dose-dependently activate DC responses in cells overexpressing CHO-K1 and CHO-claudin 18.2 ( Figure 9 IL-12 secretion and Figure 10 CD80 / CD86 expression in AB). However, the bispecific antibody can only activate DC responses in the presence of cells overexpressing claudin 18.2. And the efficacy is related to the expression level of CLDN18.2.

[0302] 5.3 Bispecific Antibodies Promote Human B Cell Immune Responses

[0303] To investigate the ability of the claudin 18.2-CD40 bispecific antibody to activate human B cells, the Ki67 (cell proliferation) and CD86 expression (cell activation) of B cells were detected. Human B cells were isolated from human PBMCs using a B cell isolation kit according to the manufacturer's protocol. Human B cells were used as effector cells. CHO-K1 cells expressing claudin 18.2 were used as target cells. Human B cells (5×10 4 ) and CHO-K1-claudin 18.2 or parental CHO-K1 cells (1×10 4 The bispecific antibodies were serially diluted 10-fold and added to the culture medium at a final concentration starting from 100 nM. After 72 hours of incubation, the Ki67 and CD86 expression of B cells were detected by FACS according to the staining and analysis procedures. Briefly, the stimulated B cells were harvested by pipetting and washed with FACS buffer. BV421 mouse anti-human CD86 and APC mouse anti-human CD19 (BD) were added to each well and incubated at 4 ° C for 30 minutes. After washing, the cells were fixed and permeabilized using Foxp3 / transcription factor staining buffer set (Invitrogen) and then Alexa Fluor 500 was added at 4 ° C. The cells were stained with AF488 anti-human Ki-67 antibody for 30 minutes. After washing, the MFI of AF488 and BV421 gated on CD19+ cells was evaluated using a MACSQuant analyzer.

[0304] like Figure 11 As shown, selenulumab monoclonal antibody can dose-dependently activate B cell responses in cells overexpressing CHO-K1 and CHO-claudin 18.2 ( Figure 11 The bispecific antibody activated B cell responses only in the presence of cells overexpressing claudin 18.2, and the efficacy was correlated with the expression level of CLDN18.2, further demonstrating the tumor antigen-dependent nature of the CD40 agonist antibody.

[0305] Example 6. Humanization of CD40 VHH Antibody

[0306] Humanized mAbs were generated using the variable region genes from 42p155 and 2p442. In the first step of this approach, the amino acid sequences of the VHHs from 42p155 and 2p442 were compared to available human Ig gene sequence databases to identify the best overall matching human germline Ig gene sequences. Humanized variable domain sequences from 42p155 and 2p442 were then designed, with CDRH1, H2, and H3 located on the framework sequences of their respective VH genes.

[0307] The amino acid sequences of the partially humanized antibodies are listed in Table 6 below.

[0308] Table 6-1. Humanized 42p155 antibody sequences (underlined CDRs; bold / italics indicate back mutations; bold indicates PTM removal)

[0309]

[0310] Table 6-1A.42p155 CDR sequences (with PTM de-risked form)

[0311]

[0312]

[0313] Table 6-2. Humanized 2p442 antibody sequences (underlined CDRs; bold / italics indicate back mutations)

[0314]

[0315] The back mutations of 42p155 include 1P, 2S, 88P, and 98Q. More specifically, VHH-v2 includes the back mutation 98Q; VHH-v3 includes the back mutations 88P and 98Q; and VHH-v4 includes the back mutations 1P, 2S, 88P, and 98Q.

[0316] The back mutations of 2p442 include 30D, 37F, 44E, 45R, 47G, 78V, 87P, and 97A. More specifically, VHH-v8 includes the back mutations 30D, 37F, 45R, 47G, 78V, and 87P; VHH-v9 includes the back mutations 30D, 37F, 44E, 47G, 78V, and 87P; and VHH-v10 includes the back mutations 30D, 37F, 44E, 47G, 78V, 87P, and 97A.

[0317] The humanized VHH genes were cloned into the pcDNA3.4 vector and transfected into 293F cells for further analysis.

[0318] Example 7: Antigen Binding Properties of Humanized Antibodies

[0319] 7.1 Full kinetic affinity of humanized antibodies measured by Biacore and Octet

[0320] The binding of humanized 42p155 and 2p442 antibodies to recombinant human CD40 protein (human CD40-his tag) was tested by Biacore and Octet using capture methods, respectively.

[0321] For the humanized 42p155 monoclonal antibodies, 42p155z2 and 42p155z3 were captured using a Protein A chip. Serial dilutions of human CD40-His-tagged protein were injected over the captured antibodies at a flow rate of 10 μl / min for 3 minutes. Antigen dissociation was allowed for 6 minutes. All experiments were performed on a Biacore T200. Data were analyzed using Biacore T200 evaluation software.

[0322] For the humanized 2p442 monoclonal antibody, 2p442z8, 2p442z9, and 2p442z10 were captured using an AHC biosensor. Serial dilutions of human CD40-his-tagged protein were incubated with the captured antibody for 5 minutes. Antigen dissociation was allowed for 10 minutes. All experiments were performed on an Octet RED96e. Data were analyzed using Octet Analysis Studio 12.2 software.

[0323] The results are shown below in Table 7. All humanized antibodies showed moderate binding and were comparable to the parent chimeric antibody.

[0324] Table 7-1 Full kinetics of humanized 42p155 measured by Biacore

[0325]

[0326] Table 7-2 Full kinetics of humanized 2p442 measured by Octet

[0327]

[0328] 7.2 Binding of Humanized 2p442 Antibody to Cell Surface CD40

[0329] To evaluate the binding affinity of humanized 2p442 monoclonal antibodies to cell surface CD40, 2p442z8, 2p442z9, 2p442z10, and parental 2p442 were tested by FACS in Jurkat cells overexpressing CD40. A total of 1 × 10 5 Jurkat-CD40 cells were incubated with 3-fold serial dilutions of antibodies starting from 100 nM at 4°C for 30 minutes. After washing with FACS buffer, PE-conjugated anti-human IgG antibodies were added to each well and incubated at 4°C for 30 minutes. After washing, the MFI of PE was evaluated by MACSQuant analyzer16. Figure 12 As shown, the humanized 2p442 antibody displayed comparable binding activity to the parent chimeric antibody.

[0330] Example 8. Functional activity of humanized bispecific antibodies

[0331] Anti-Claudin 18.2 / CD40 bispecific antibodies were prepared using humanized CD40 nanobodies and tested in this example. 2p834z2, 2p834z3, and 2p834z4 used the same human FRs as 42p155z2, 42p155z3, and 42p155z4, respectively, to generate humanized Claudin 18.2-2p-834 bispecific antibodies. The amino acid sequences of the humanized 2p834 antibodies are listed in Table 8 below.

[0332] Table 8. Humanized antibody sequences (underlined CDRs; bold / italics indicate back mutations)

[0333]

[0334] 8.1 Cell Line-Based Functional Characterization of a Humanized Claudin 18.2-CD40 Bispecific Antibody

[0335] To evaluate the ability of humanized tight junction protein 18.2-CD40 bispecific antibodies to activate the CD40 signaling pathway, a commercial CD40 NFκB luciferase reporter gene system was used, as described in Example 5.1. Briefly, H_CD40 (TNFRSF5) NFκB-reporter Jurkat cells were used as effector cells, and cells expressing CHO-K1 or not expressing tight junction protein 18.2 were used as target cells. In a white 96-well plate, effector cells and target cells were co-cultured with an E / T ratio of 1:1. The antibody was serially diluted 5 times and added to a white 96-well assay plate at a final concentration range of 0.001nM to 100nM. Luminescence was obtained after incubation for 5 hours by adding luciferase substrate and measured by a microplate reader.

[0336] like Figure 13 As shown, the humanized anti-CLDN18.2 / CD40 bispecific antibody showed considerable activity in inducing the CD40 signaling pathway.

[0337] 8.2 Humanized Bispecific Antibodies Promote the Activity of Human Dendritic Cell Immune Responses

[0338] To investigate the ability of the humanized CLDN18.2 / CD40 bispecific antibody to stimulate human dendritic cell (DC) responses, IL-12 cytokine release and CD80 / CD86 expression by DC were measured, as described in Example 5 above. Human DC were obtained according to the procedure specified in Example 3.1. Human DC were used as effector cells. CHO-K1 cells expressing claudin 18.2 were used as target cells. Human DC (5×10 4 ) and CHO-K1-claudin 18.2 or parental CHO-K1 cells (1.5×10 4 Humanized bispecific antibodies were added to the culture medium at a final concentration starting from 100 nM. After 48 hours of incubation, IL-12 levels in the culture medium were measured using the IL-12 / p40 (human) LANCE Ultra TR-FRET Detection Kit (PerkinElmer). In addition, DCs were tested for CD80 / CD86 expression by FACS according to the staining and analysis procedures described in Example 5.2. Figure 14 and Figure 15 As shown, humanized bispecific antibodies induced DC responses ( Figure 14 IL-12 secretion and Figure 15 CD80 / CD86 expression in A / B) was comparable to their chimeric antibodies.

[0339] 8.3 Humanized Bispecific Antibodies Promote Human B Cell Immune Responses

[0340] To investigate the ability of the humanized claudin 18.2-CD40 bispecific antibody to activate human B cells, the expression of Ki67 (cell proliferation) and CD86 on B cells was examined. As described in Example 5, human B cells were isolated from human PBMCs using a B cell isolation kit according to the manufacturer's protocol. Human B cells were used as effector cells. CHO-K1 cells expressing claudin 18.2 were used as target cells. Human B cells (5×10 4 ) and CHO-K1-claudin 18.2 or parental CHO-K1 cells (1×10 4 ) (E / T ratio = 5:1). 5-fold serial dilutions of the humanized bispecific antibody were added to the culture medium at a final concentration starting from 100 nM. After incubation for 72 hours, the Ki67 / CD86 expression of B cells was detected by FACS according to the staining and analysis procedures described in Example 5.3. Figure 16 As shown, the B cell response induced by humanized bispecific antibodies ( Figure 16 Ki67 / CD86 expression in A / B) were comparable to their chimeric antibodies.

[0341] Example 9. Anti-Claudin 18.2 / CD40 Bispecific Antibody Inhibits Tumor Growth

[0342] In this example, humanized mice in which the extracellular domain of mouse CD40 was replaced with the corresponding human CD40 were used to test the anti-tumor activity of bispecific antibodies.

[0343] Mouse colon adenocarcinoma cells (MC38) were engineered to express human CLDN18.2. MC38-hCLDND18.2 cells were subcutaneously implanted into humanized C57bl / 6 mice (huCD40). Figure 17 As shown in A, the following antibodies were administered intraperitoneally to mice twice a week for a total of 6 times: human IgG control (3.6 mg / kg), serotonin (3 mg / kg), 42p155 anti-CLDN18.2 / CD40 bispecific antibody (3.6 mg / kg, equimolar to serotonin), and 2p834 anti-CLDN18.2 / CD40 bispecific antibody (3.6 mg / kg, equimolar to serotonin).

[0344] like Figure 17 As shown in B, all mice treated with 42p155 anti-CLDN18.2 / CD40 biAb or 2p834 anti-CLDN18.2 / CD40 biAb had complete tumor regression, while mice treated with selumab showed only a moderate response. In addition, 51 days after the first treatment, MC38-hCLDND18.2 cells were injected subcutaneously into the contralateral flank of tumor-free mice (at Figure 17Indicated by black arrows in B) to determine the formation of immune cell memory against this tumor cell line. C57bl / 6-huCD40 mice were injected with the same MC38-hCLDND18.2 tumor cells. Tumor volume was monitored by caliper measurement twice a week during the experiment.

[0345] like Figure 17 As shown in Table 9 and Table 1, selenumab inhibited tumor growth with a TGI of 78.3% on day 23 after the first treatment. Meanwhile, the 42p155 and 2p834 bispecific antibodies induced complete tumor remission in all treated mice starting on day 27. Furthermore, after rechallenge, no MC38-hCLDND18.2 tumors grew in the groups previously treated with the 42p155 and 2p834 bispecific antibodies, while 100% of tumors grew in naive mice. These results demonstrate that CD40-targeted therapy has strong anti-tumor efficacy and tumor growth regression compared to non-targeted CD40 therapy. Rechallenge data indicate that an effective immune memory response against MC38-hCLDND18.2 tumor cells was formed in all mice treated with the 42p155 or 2p834 bispecific antibodies.

[0346] Table 9. TGI of the MC38-CLDN18.2 mouse model.

[0347] Group TGI of D23 (%) Selumetumab 78.3％ 42p155BiAb 103.2％ 2p834BiAb 102.8％

[0348] To evaluate immune activation by CD40 therapy, tumor-infiltrating immunophenotyping (IPT) analysis was performed on day 7 after intraperitoneal administration and twice weekly for a total of 2 ( Figure 18 A). Figure 18 As shown in CF, the 42p155 bispecific antibody induced significant immune responses in tumor tissues, including the expression of leukocytes, T cells (CD8 + and CD4 + In contrast, selumetab only slightly increased T cells, especially CD8 + In addition, compared with the PBS and selumetab groups, the 42p155-BiAb also enhanced the proliferation of CD8 T cells, CD4 T cells, and B cells, and increased the expression of CD80 and CD86 in DCs and B cells. These data suggest that the 42p155 bispecific antibody can significantly enhance the immune response in tumor tissues, outperforming CD40 agonist monoclonal antibodies.

[0349] To determine whether CD40 therapy had any effect on the peripheral immune system, immunophenotyping (IPT) analysis of the spleen was also performed on day 7. Figure 18 As shown in the GI, 42p155-BiAb did not significantly affect peripheral immune cell populations, whereas selukumab reduced T cells. Simultaneously, activated B cells also increased after selukumab treatment. These data suggest that selukumab activates peripheral CD40, whereas 42p155-BiAb remains silent in the periphery.

[0350] To evaluate the toxicity of CD40 activation on liver function, the AST and ALT concentrations in the blood were measured on day 20 after intraperitoneal administration and twice a week for a total of 6 times ( Figure 18 A). Figure 18 As shown in Figure B, the 42p155 bispecific antibody did not increase ALT or AST levels compared with the PBS group, whereas the selumetumab-treated group increased ALT and AST levels. These data suggest that the 42p155 bispecific antibody may minimize the risk of peripheral toxicity by concentrating CD40 activation primarily in the tumor environment.

[0351] Example 10: Generation of mouse monoclonal antibodies against human 5T4

[0352] This example describes the generation of anti-human 5T4 mouse monoclonal antibodies using hybridoma technology.

[0353] Antigens: human 5T4-His protein and human 5T4 expressing CHO-K1 (CHOK1-hu5T4).

[0354] Immunization: To generate mouse monoclonal antibodies targeting human 5T4, SJL, Balb / C, and C57BL / 6 mice were first immunized with 5T4-His protein. Immunized mice were subsequently boosted with 5T4-His protein or human 5T4 expressing CHO-K1. To select mice producing antibodies that bind to 5T4 protein, sera from immunized mice were evaluated for antibody titers by ELISA and FACS. Briefly, microtiter plates were coated with 100 μL / well of 0.5 μg / mL or 1 μg / mL human 5T4 or cynomolgus monkey 5T4 protein in ELISA coating buffer overnight at 4°C and then blocked with 150 μL / well of 1% BSA. Dilutions of serum from immunized mice were added to each well and incubated at 37°C for 1 hour. The plates were washed with PBS / Tween and then incubated with anti-mouse IgG antibody conjugated to horseradish peroxidase (HRP) at 37°C for 30 minutes. After washing, the plates were developed with TMB substrate and analyzed spectrophotometrically at OD 450 nm. The immune response to the CHOK1-hu5T4 cell line was also tested by serum FACS, with the CHOK1 parental cell line used as a negative control. The resulting mice were used for fusion. Hybridoma supernatants were screened by ELISA.

[0355] Cell fusion: Fusion was performed by electrofusion. The fused cells were seeded into 50 96-well plates for each fusion.

[0356] Screening: Hybridoma supernatants were screened by ELISA against recombinant human (rh) 5T4-His protein and recombinant cynomolgus 5T4-His protein. Positive supernatants from the primary screen were then confirmed by FACS binding to CHOK1-hu5T4 cell lines and ELISA binding protein.

[0357] Subcloning and screening: Positive primary clones from each fusion were subcloned by limiting dilution to ensure that the subclones were derived from a single parental cell. Subclones were screened in the same manner as the primary clones, and culture supernatants of positive clones were subjected to additional confirmatory screening by affinity ranking.

[0358] Clones 14G12, 393E9, 159D5, and 286B4 were selected for further analysis and humanization. The 5T4 mAbs were classified into four segments (Segment A, Segment B, Segment C, and Segment D) based on the binding epitope on human 5T4 by competitive ELISA. As a reference, the Fab portion of naptumomab also binds to Segment A of 5T4. 14G12 and 393E9 belong to Segment A, 159D5 belongs to Segment B, and 286B4 belongs to Segment D.

[0359] Example 11. Humanization of 5T4 antibodies

[0360] Variable region genes were employed to generate humanized mAbs. In the first step of this process, the amino acid sequences of the VH and VK were compared to available databases of human Ig gene sequences to find the overall best matching human germline Ig gene sequences.

[0361] The sequences of the human germlines used for CDR grafting, as well as the resulting humanized sequences, are listed in Table 10.

[0362] Table 10-1. Humanization of 14G12 (underlined = CDR; bold / italic = back mutations)

[0363]

[0364]

[0365] Table 10-2. Humanized antibodies from 14G12

[0366]

[0367] Table 10-3. Humanization of 393E9 (underlined = CDR; bold / italic = back mutations)

[0368]

[0369]

[0370] Table 10-4. Humanized antibodies from 393E9

[0371]

[0372]

[0373] Table 10-5. Humanization of 159D5 (underlined CDRs; bold / italics indicate back mutations)

[0374]

[0375]

[0376] Table 10-6. Humanized antibodies from 159D5

[0377]

[0378] Table 10-7. Humanization of 286B4 (underlined CDRs; bold / italics indicate back mutations, and boxes indicate potential PTM site removals)

[0379]

[0380]

[0381] Table 10-8. Humanized antibodies from 286B4

[0382]

[0383] Example 12. Selection of the anti-5T4 portion of the 5T4-CD40 bispecific antibody

[0384] This example tests the functional activity of the 5T4-CD40 bispecific antibody and selects the 5T4 portion of the 5T4-CD40 bispecific antibody.

[0385] Generation of 5T4-CD40 bispecific antibodies with different 5T4 binding epitopes

[0386] like Figure 4The 5T4 sequences of 14G12, 393E9, 159D5, and 286B4 were constructed into 5T4 moieties in the "2+2bll" format as shown. 14G12 and 393E9 belong to segment A, 159D5 belongs to segment B, and 286B4 belongs to segment D. The CD40 sequence of 42p155 was used as the anti-CD40 moiety. The peptide chain of the "2+2bll" format bispecific antibodies is shown in Tables 11A-D.

[0387] Table 11A. Peptide chains of 14G12-42p155 bispecific antibodies

[0388]

[0389] Table 11B. Peptide chains of 393E9-42p155 bispecific antibodies

[0390]

[0391]

[0392] Table 11C. Peptide chains of 159D5-42p155 bispecific antibodies

[0393]

[0394] Table 11D. Peptide chains of 286B4-42p155 bispecific antibodies

[0395]

[0396] Cell line-based functional characterization of 5T4-CD40 bispecific antibodies with different 5T4 binding epitopes

[0397] To assess the ability of 5T4-CD40 bispecific antibodies to activate the CD40 signaling pathway, a commercial CD40 NF-κΒ luciferase reporter system was used. In this assay, H_CD40 (TNFRSF5) NFκΒ-reporter Jurkat (Genomeditech, cat# GM-C09520) was used as effector cells, and cells expressing human 5T4 (MCF-7: 5T4 低 and CHO-K1-hu5T4: 5T4 高 ) or non-expressing cells (CHO-K1) were used as target cells. Briefly, effector cells at a density of 2.0 x 10 4 cells / well were mixed with 2.0 x 10 4Target cells (E / T ratio = 1:1) were co-cultured in a white 96-well plate. Antibodies were serially diluted 10-fold and added to a white 96-well assay plate at a final concentration range of 0.001 nM to 100 nM. After incubation at 37°C for 5 hours, luminescence was obtained by adding luciferase substrate and measured by a microplate reader. Four-parameter logistic curve analysis was performed using GraphPad software.

[0398] like Figure 19 As shown, the activities of the tested 5T4-CD40 bispecific antibodies with different epitopes in the 5T4 protein were solely or partially dependent on the expression of hu5T4 in cells, indicating that activation of CD40 signaling was much stronger in the presence of hu5T4. In addition, the 5T4-CD40 bispecific antibodies with both segments A and B of the 5T4 portion showed strong agonist activity, especially when the target cells expressed moderate levels of hu5T4 on their surface.

[0399] 5T4-CD40 bispecific antibody promotes the activity of human dendritic cell immune response

[0400] To investigate the ability of 5T4-CD40 bispecific antibody to stimulate human dendritic cell (DC) responses, IL-12 cytokine release and CD80 / CD86 expression by DCs were examined.

[0401] Human DCs were obtained according to the procedure specified in Example 4. Human DCs were used as effector cells. Human 5T4-expressing cells (MCF-7: 5T4 低 and CHO-K1-hu5T4:5T4 高 ) or non-expressing cells (CHO-K1) were used as target cells. Human DCs (5×10 4 ) and target cells (1.5×10 4 ) co-cultured (E / T ratio = approximately 3:1). The bispecific antibody was serially diluted 10-fold and added to the culture medium at a final concentration starting at 100 nM. After 48 hours of incubation, IL-12 levels in the culture medium were measured using the IL-12 / p40 (human) LANCE Ultra TR-FRET Detection Kit (PerkinElmer). Data were analyzed using nonlinear regression, a 4-parameter logistic equation.

[0402] Activation of DCs leads to upregulation of the costimulatory molecules CD80 / CD86. Here, CD80 / CD86 expression on DCs was detected by FACS according to the staining and analysis procedure. Briefly, stimulated DCs were harvested by pipetting and washed with FACS buffer. After blocking with FcR blocking reagent (MACS) for 15 min at 4°C, PE mouse anti-human CD80, BV421 mouse anti-human CD86, and APC mouse anti-human CDl lc (BD) were added to each well and incubated for 30 min at 4°C. After washing, the MFI of PE and BV421 gated on CDl lc+ cells was evaluated by MACSQuant Analyzer 16.

[0403] As shown in Figure 20 and Figure 21 , 5T4-CD40 bispecific antibodies only activated the DC response in the presence of hu5T4-expressing cells. And the potency correlated with the expression level of hu5T4. Similarly, 5T4-CD40 bispecific antibodies with segment A and segment B of the 5T4 moiety showed stronger agonist activity.

[0404] Example 13. Generation of bispecific antibodies with different bispecific antibody formats

[0405] To further activate the CD40 pathway by 5T4-CD40 bispecific antibodies, we designed several bispecific antibody formats to determine the optimal format for 5T4-CD40. The configuration of the bispecific antibodies for each mode of specificity to a single target is shown in Figure 22 The peptide chains of the bispecific antibodies of different formats are shown in Table 12A, and the sequences of each target used in the format are shown in Tables 12B-C.

[0406] Table 12A. Peptide chains of exemplary bispecific antibodies

[0407]

[0408]

[0409]

[0410]

[0411]

[0412]

[0413]

[0414] Table 12B. Antibody variable region sequences of CD40 moiety

[0415]

[0416]

[0417] Table 12C. Antibody variable region sequences of the 5T4 portion used / suitable for use in bispecific antibodies

[0418]

[0419] Example 14. Selection of 5T4-CD40 formats with different CD40 activities

[0420] To select a suitable format for the 5T4-CD40 bispecific antibody, we tested the functional activity of 5T4-CD40 bispecific antibodies with different formats and CD40 activities to select the most potent and cleanest combination of 5T4-CD40 bispecific antibodies.

[0421] Binding activity of bispecific antibodies to human CD40 on cell surfaces

[0422] To evaluate the binding affinity of 5T4-CD40 bispecific antibodies to cell surface CD40, bispecific antibodies with the formats b11 (42p155z2), b12 (42p155z2), b13 (42p155z2), b16 (42p155z2), b17 (42p155z2), and b18 (2p1130) were tested by FACS in Jurkat cell lines overexpressing CD40. A total of 1×10 5 Jurkat-CD40 cells were incubated with 5-fold serial dilutions of antibodies starting from 100 nM at 4°C for 30 minutes. After washing with FACS buffer, PE-conjugated anti-human IgG antibodies were added to each well and incubated at 4°C for 30 minutes. After washing, the MFI of PE was evaluated by MACSQuant analyzer16. Figure 23 As shown in Figure 2, the binding activity of the tested 5T4-CD40 bispecific antibodies was dependent on the antibody format as well as the CD40 clone. Figure 29 As shown, the humanized version of the 5T4-CD40 biAb, b16(42p155z2)-LALA, displayed comparable binding to its chimeric antibody b16(42p155z2) against CD40 expressed on DCs.

[0423] Cell line-based functional characterization of 5T4-CD40 bispecific antibodies with different formats and CD40 clones

[0424] To evaluate the efficacy of the 5T4-CD40 bispecific antibody in activating the CD40 signaling pathway, a commercial CD40 NF-κB luciferase reporter gene system was used. In this assay, H_CD40 (TNFRSF5) NFκB-reporter Jurkat (Genomeditech, cat# GM-C09520) was used as the effector cell, and cells expressing human 5T4 (MCF-7: 5T4 低 and CHO-K1-hu5T4:5T4 高 ) or non-expressing cells (CHO-K1) were used as target cells. Briefly, cells were plated at a density of 2.0 × 10 4 Effector cells / well and 2.0×10 4 Target cells (E / T ratio = 1:1) were co-cultured in a white 96-well plate. Antibodies were serially diluted 10-fold and added to a white 96-well assay plate at a final concentration range of 0.001 nM to 100 nM. After incubation at 37°C for 5 hours, luminescence was obtained by adding luciferase substrate and measured by a microplate reader. Four-parameter logistic curve analysis was performed using GraphPad software.

[0425] like Figure 24 As shown, the activities of the tested 5T4-CD40 bispecific antibodies with different formats and CD40 clones were solely or partially dependent on the expression of hu5T4 in cells, indicating that the activation of CD40 signaling was much stronger in the presence of hu5T4. In addition, the activities of the tested 5T4-CD40 bispecific antibodies were significantly different, and the bispecific antibodies with b16 and b18 formats showed much stronger activities than the other formats. In addition, as Figure 30 As shown, when expressed with 5T4, the humanized form of the 5T4-CD40 biAb, b16(42p155z2)-LALA, displayed comparable activity to its chimeric antibody b16(42p155z2) in inducing NF-κB signaling downstream of CD40.

[0426] 5T4-CD40 bispecific antibody promotes the activity of human dendritic cell immune response

[0427] To investigate the ability of the 5T4-CD40 bispecific antibody to stimulate human dendritic cell (DC) responses, IL-12 cytokine release from DCs was examined.

[0428] Human DCs were obtained according to the procedure specified in Example 4. Human DCs were used as effector cells. Human 5T4-expressing cells (MCF-7: 5T4 低 and CHO-K1-hu5T4:5T4 高 ) or non-expressing cells (CHO-K1) were used as target cells. Human DCs (5×104 ) and target cells (1.5×10 4 ) co-cultured (E / T ratio = approximately 3:1). The bispecific antibody was serially diluted 10-fold and added to the culture medium at a final concentration starting at 100 nM. After 48 hours of incubation, IL-12 levels in the culture medium were measured using the IL-12 / p40 (human) LANCE Ultra TR-FRET Detection Kit (PerkinElmer). Data were analyzed using nonlinear regression, a 4-parameter logistic equation.

[0429] like Figure 25 As shown in Figure 2, the activity of the tested 5T4-CD40 bispecific antibodies with different formats and CD40 clones was solely or partially dependent on the expression of hu5T4 in the cells, indicating that the activation of CD40 signaling was much stronger in the presence of hu5T4. Similarly, the potency of the tested 5T4-CD40 bispecific antibodies varied significantly, and the bispecific antibodies with b16 and b18 formats showed much stronger agonist activity than the other antibodies. In addition, as Figure 31 As shown, when expressed with 5T4, the humanized form of 5T4-CD40 biAb, b16(42p155z2)-LALA, displayed comparable activity to its chimeric antibody b16(42p155z2) in inducing DCs to secrete IL2.

[0430] Example 15.5 Inhibition of Tumor Growth by T4-CD40 Bispecific Antibody

[0431] In this example, humanized mice in which the extracellular domain of mouse CD40 was replaced with its human counterpart CD40 were used to test the anti-tumor activity of the 5T4-CD40 bispecific antibody.

[0432] Mouse colon adenocarcinoma cells (MC38) were engineered to express human 5T4 (MC38-hu5T4). MC38-hu5T4 cells were implanted subcutaneously into humanized C57BL / 6 mice (huCD40). Figure 26 As shown in A, mice were intraperitoneally administered with the following antibodies twice a week for a total of 4 times: PBS control, selenumab (3 mg / kg), b18(2p1130) (3 mg / kg), b18(2p1130) (10 mg / kg), b16(2p1130) (2.5 mg / kg) and b16(42p155z2) (2.5 mg / kg). All antibodies tested were administered in equimolar amounts.

[0433] like Figure 26As shown in Table B and Table 13, mice treated with selumab and b16(2p1130) showed only a moderate response. On day 25, selumab inhibited tumor growth with a TGI of 76.1%, and b16(2p1130) inhibited tumor growth with a TGI of 83.8%. At the same time, b18(2p1130) (3 mg / kg) and b16(42p155z2) induced complete tumor remission in 4 / 6 mice and 5 / 6 mice, respectively, from day 39. And all mice treated with b18(2p1130) (10 mg / kg) had complete tumor regression from day 35. These results indicate that 5T4-targeted CD40 therapy has strong anti-tumor efficacy and tumor growth regression compared to non-targeted CD40 therapy. To determine whether CD40-targeted therapy can induce immune memory, a second challenge of MC38-hu5T4 was administered to the contralateral flank of mice with complete tumor remission. As Figure 26 As shown in C, all of these mice were resistant to tumor re-challenge, indicating that long-lasting immune memory responses were established in mice treated with the 5T4-CD40 bispecific antibodies b18(2p1130) and b16(42p155z2).

[0434] Immunophenotyping (IPT) analysis in peripheral blood was performed on day 7 after the first administration. Figure 27 As shown, b16(2p1130) and b16(42p155z2) did not change the cell counts of B cells compared with the PBS group, whereas selumetumab and b18(2p1130) decreased the cell number ( Figure 27 A). In addition, selumetumab significantly increased the expression of CD80 and CD86 in B cells and the proliferation of T cells, whereas other bispecific antibodies did not change or only slightly increased activation markers ( Figure 27 BC). These data suggest that b16(2p1130) and b16(42p155z2) do not activate immune cells in the periphery.

[0435] To evaluate immune activation based on CD40 therapy, tumor infiltration IPT analysis was performed on day 7 in a separate group with the same experimental design ( Figure 28 A). Figure 28 As shown in Figures BE, b16(42p155z2) induced immune responses in tumor tissues, including increased percentages of leukocytes, CD4+ T cells, and DCs compared with the PBS group. Furthermore, b16(42p155z2) also increased the expression of CD80 and CD86 in DCs compared with the PBS group. These data suggest that the 5T4-CD40 bispecific antibody is a promising therapeutic approach for treating 5T4-expressing tumors through 5T4-dependent CD40 activation.

[0436] Table 13. TGI in MC38-hu5T4 mouse model

[0437] Group TGI of D25 (%) Selumetinib (3mpk) 76.1％ b18(2p1130)(3mpk) 105.3％ b18(2p1130)(10mpk) 105.2％ b16(2p1130)(2.5mpk) 83.8％ b16(42p155z2)(2.5mpk) 94.2％

[0438] ***

[0439] The scope of the disclosure is not intended to be limited to the particular embodiments described herein, as such can vary. It is therefore understood that within the scope of the disclosure, modifications will occur to those skilled in the art to which the disclosure pertains, and which are intended to be within the scope of the claims and equivalents thereof. It is further understood that where particular embodiments are described, these are only by way of example, and that other embodiments are contemplated.

[0440] All publications and patent applications mentioned in the specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Claims

1. A bispecific antibody comprising a first antibody or antigen-binding fragment having binding specificity for a CD40 protein, and a second antibody or antigen-binding fragment having binding specificity for a 5T4 protein, wherein the bispecific antibody more effectively activates CD40 on target cells expressing the 5T4 protein than on reference cells not expressing the 5T4 protein, or wherein the bispecific antibody does not activate CD40 on reference cells not expressing the 5T4 protein; wherein the first antibody or antigen-binding fragment comprises two tandem single-domain (VHH) anti-CD40 antibodies, each of the single-domain (VHH) anti-CD40 antibodies comprising: a CDR1 having an amino acid sequence of SEQ ID NO: 14, a CDR2 having an amino acid sequence of SEQ ID NO: 15, 63 or 64, and a CDR3 having an amino acid sequence of SEQ ID NO: 16; wherein the second antibody or antigen-binding fragment thereof comprises a conventional VH / VL Fab fragment, and comprises a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region comprises VH CDR1, VH CDR2 and VH CDR3, the light chain variable region comprises VL CDR1, VL CDR2 and VL CDR3, wherein the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VLCDR2 and VL CDR3 are the amino acid sequences of SEQ ID NOs: 75-80, respectively; The two tandem single domain (VHH) anti-CD40 antibodies and the conventional VH / VL Fab fragments of the second antibody are fused to the N-terminus of each of the two chains of the Fc fragment. The bispecific antibody according to claim 1 , wherein the Fc fragment is a human IgG1, IgG2 or IgG4 fragment.

3. The bispecific antibody of claim 2, wherein the Fc fragment comprises L234A, L235A and N297A, L234A and L235A, or N297A substitutions according to Kabat numbering.

4. The bispecific antibody of claim 1, wherein each of the VHH antibodies comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 53-62.

5. The bispecific antibody of claim 1, wherein each of the VHH antibodies comprises the amino acid sequence of SEQ ID NO:

54.

6. The bispecific antibody according to any one of claims 1 to 5, wherein the second antibody or antigen-binding fragment competes with antibody 14G12 for binding to the 5T4 protein, wherein the antibody 14G12 comprises the VH of SEQ ID NO: 73 and the VL of SEQ ID NO:

74.

7. The bispecific antibody of claim 1, wherein the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 73 and 81-90, and the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 74 and 91-100.

8. The bispecific antibody of claim 1, wherein the VH comprises the amino acid sequence of SEQ ID NO: 83 or 89, and the VL comprises the amino acid sequence of SEQ ID NO:

91.

9. A 5T4-CD40 bispecific antibody comprising a heavy chain (VH) and a light chain (VL), wherein the amino acid sequence of the VH is SEQ ID NOs: 73, 158, 159, and 54 connected end-to-end from the N-terminus to the C-terminus, and the amino acid sequence of the VL is SEQ ID NOs: 74 and 160 connected end-to-end from the N-terminus to the C-terminus.

10. A 5T4-CD40 bispecific antibody, comprising a heavy chain 1 (VH1), a light chain (VL), and a heavy chain 2 (VH2), wherein the amino acid sequence of the VH1 is SEQ ID NOs: 73 and 161, which are connected end to end from the N-terminus to the C-terminus, the amino acid sequence of the VL is SEQ ID NOs: 74 and 160, which are connected end to end from the N-terminus to the C-terminus, and the amino acid sequence of the VH2 is SEQ ID NOs: 54 and 162, which are connected end to end from the N-terminus to the C-terminus.

11. A 5T4-CD40 bispecific antibody comprising a heavy chain (VH) and a light chain (VL), wherein the amino acid sequence of the VH is SEQ ID NO: 73 and 158 connected end to end from the N-terminus to the C-terminus, and the amino acid sequence of the VL is SEQ ID NO: 74, 160, 159 and 54 connected end to end from the N-terminus to the C-terminus.

12. A 5T4-CD40 bispecific antibody comprising a heavy chain 1 (VH1), a light chain (VL), and a heavy chain 2 (VH2), wherein the amino acid sequence of the VH1 is SEQ ID NO: 73, 161, 159, and 54 are linked end-to-end from the N-terminus to the C-terminus, the amino acid sequence of the VL is SEQ ID NO: 74 and 160 are linked end-to-end from the N-terminus to the C-terminus, and the amino acid sequence of the VH2 is SEQ ID NO: 162, 159, and 54 are linked end-to-end from the N-terminus to the C-terminus.

13. A 5T4-CD40 bispecific antibody comprising a heavy chain 1 (VH1), a light chain (VL), and a heavy chain (VH2), wherein the amino acid sequence of the VH1 is SEQ ID NOs: 73 and 161 connected end to end from the N-terminus to the C-terminus, the amino acid sequence of the VL is SEQ ID NOs: 74 and 160 connected end to end from the N-terminus to the C-terminus, and the amino acid sequence of the VH2 is SEQ ID NOs: 13, 163, 13, 163, 13, and 162 connected end to end from the N-terminus to the C-terminus.

14. One or more polynucleotides encoding the bispecific antibody according to any one of claims 1 to 13.

15. The polynucleotide of claim 14, which is one or more mRNAs, wherein the mRNAs are chemically modified.

16. A cell comprising the polynucleotide of claim 15.

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