Tumor antigen-dependent CD40 agonist single-domain antibody

Abstract

The present invention provides CD40 agonist single domain antibodies that have significantly higher CD40 activation activity in the presence of simultaneous tumor-associated antigen (TAA) binding compared to the situation without 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 bispecific and multispecific antibodies and polypeptides, such as chimeric antigen receptors, which are optionally incorporated into these antibodies together with anti-TAA units.

Description

[0001] This application is a divisional application of a patent application for invention titled "Tumor Antigen-Dependent CD40 Agonist Antibody", with the application number 202380030002.2, the filing date of February 21, 2023, and which entered the Chinese national phase on September 24, 2024. Background Art

[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 co-stimulatory 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 regulating the activity of APCs and linking innate immunity and adaptive immunity. The CD40 pathway is not only required for effective T cell and B cell immune responses but also provides a crucial initial step in the development of 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 the 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, it has been reported that CD40-activated macrophages rapidly infiltrate tumors and promote the depletion of the tumor stroma, and further enhance chemotherapy delivery. In summary, CD40 activation contributes to an important mechanism for converting so-called cold tumors into hot tumors.

[0004] Multiple methods have been developed to activate CD40 in cancer patients. The initial CD40 therapeutic agonists were based on multimeric forms of its ligand, i.e., CD40L. Subsequently, the approach has mainly been based on agonist CD40 antibodies, which are 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 other antibodies (e.g., selicrelumab and CDX-1140) do not block.

[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 a variety of combinations of chills, rigors, rash, nausea, fever, vomiting, myalgia, and back pain. Another major safety concern is dose-related hematotoxicity, such as decreases in peripheral lymphocytes, monocytes, and platelets. Such reported toxicities are considered to be legacy issues of CD40 agonist therapies 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 associated toxicities. SUMMARY OF THE INVENTION

[0006] As provided, 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 mechanism of CD40 agonism, and thus it is challenging to control these adverse effects without sacrificing therapeutic efficacy.

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

[0008] Accordingly, in one embodiment of the present disclosure, there is provided a single-domain antibody or a polypeptide comprising the single-domain antibody, wherein the single-domain antibody has binding specificity for 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 respectively comprise (1) the amino acid sequences of SEQ ID NO: 14, 15, and 16; (1a) the amino acid sequences of SEQ ID NO: 14, 63, and 16; (1b) the amino acid sequences of SEQ ID NO: 14, 64, and 16; (2) the amino acid sequences of SEQ ID NO: 17, 18, and 19; (3) the amino acid sequences of SEQ ID NO: 20, 21, and 22; (4) the amino acid sequences of SEQ ID NO: 23, 24, and 25; (5) the amino acid sequences of SEQ ID NO: 26, 27, and 28; (6) the amino acid sequences of SEQ ID NO: 29, 30, and 31; (7) the amino acid sequences of SEQ ID NO: 32, 33, and 34; (8) the amino acid sequences of SEQ ID NO: 35, 36, and 37; (9) the amino acid sequences of SEQ ID NO: 38, 39, and 40; (10) the amino acid sequences of SEQ ID NO: 41, 42, and 43; (11) the amino acid sequences of SEQ ID NO: 44, 45, and 46; (12) the amino acid sequences of SEQ ID NO: 47, 48, and 49; or (13) the amino acid sequences 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 NO: 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 NO:56, 59, and 62.

[0014] In one embodiment, provided is also a multispecific antibody that comprises an antibody of the present disclosure and a second antibody or antigen-binding fragment that has 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 that comprises a first antibody or antigen-binding fragment that has binding specificity for human CD40 and a second antibody or antigen-binding fragment that has 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 TAA as compared to CD40 on reference cells that do not express TAA.

[0016] Another embodiment provides a multispecific antibody that comprises a first antibody or antigen-binding fragment that has binding specificity for human CD40 and a second antibody or antigen-binding fragment that has binding specificity for a second target antigen, which 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 TAA at least 2-fold, or 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold the effect on CD40 on reference cells that do not express TAA. In some embodiments, the activation is measured at a concentration of the multispecific antibody of 0.001 nM to 200 nM, preferably 0.1 nM to 100 nM. In some embodiments, the activation is measured using a set 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 a conditionally activated CD40 bispecific antibody that is activated only in the presence of tumor cells expressing 5T4. 5T4 is a carcinoembryonic protein that is rarely expressed in normal adult tissues; however, its expression is upregulated in a variety of cancers. CD40 crosslinking via engagement of 5T4 on cancer cells is expected to enhance the immune response in the tumor microenvironment while minimizing the risk of peripheral toxicity. At the same time, by restricting the antibody to tumor cells expressing 5T4, the 5T4xCD40 bispecific antibody is expected to overcome antigen silencing due to widespread peripheral CD40 expression, thus allowing the molecule to accumulate in the tumor.

[0019] According to one embodiment of the present disclosure, provided is a multispecific 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 multispecific antibody more effectively activates CD40 on target cells expressing the 5T4 protein as compared to CD40 on a reference cell that does not express the 5T4 protein, or wherein the multispecific antibody does not activate CD40 on a reference cell that does not express the 5T4 protein.

[0020] In some embodiments, the multispecific antibody activates CD40 on target cells expressing the 5T4 protein at least 2-fold, or 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold more effectively than CD40 on a reference cell that does not express the 5T4 protein. In some embodiments, the activation is measured with 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 with a panel of CD40 functional assays; preferably, wherein the activation is measured with an NFκB reporter 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.

[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 substitutions L234A, L235A, and N297A, L234A and L235A, or N297A according to the 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, these VHH antibodies each comprise an amino acid sequence selected from SEQ ID NO:1 and 53-62. In some embodiments, these VHH antibodies each comprise the amino acid sequence of SEQ ID NO:54.

[0025] In some embodiments, the second antibody or antigen-binding fragment competes with antibody 14G12 or 159D5 for binding to the 5T4 protein, wherein antibody 14G12 comprises a VH of SEQ ID NO:73 and a VL of SEQ ID NO:74, and antibody 159D5 comprises a VH of SEQ ID NO:121 and a 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, and the light-chain variable region comprising VL CDR1, VL CDR2, and VL CDR3, wherein the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 comprise the amino acid sequences of SEQ ID NO:130-80, respectively. 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 comprises VH CDR1, VH CDR2, and VH CDR3, and the light chain variable region comprises VL CDR1, VL CDR2, and VL CDR3, wherein the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 comprise the amino acid sequences of SEQ ID NOs: 103-108, respectively. In some embodiments, the VH comprises an amino acid sequence selected from SEQ ID NOs: 101 and 109-115, and the VL comprises an amino acid sequence selected from SEQ ID NOs: 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 comprises VH CDR1, VH CDR2, and VH CDR3, and the light chain variable region comprises VL CDR1, VL CDR2, and VL CDR3, wherein the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 comprise the amino acid sequences of SEQ ID NOs: 123-128, respectively. In some embodiments, the VH comprises an amino acid sequence selected from SEQ ID NOs: 121 and 129-131, and the VL comprises an amino acid sequence selected from SEQ ID NOs: 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] Compositions, methods, and uses for treating diseases are also provided. In one embodiment, it is 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 Shows the binding activity of an anti-CD40 monospecific antibody to human CD40 antigen and cynomolgus monkey CD40 antigen.

[0031] Figure 2 Shows the binding activity of an anti-CD40 monospecific antibody to Jurkat cells overexpressing human CD40.

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

[0033] Figure 4 Shows the format of the Claudin 18.2 / CD40 bispecific antibody.

[0034] Figure 5 Shows that the anti-Claudin 18.2 / CD40 bispecific antibody binds to human dendritic cells with an activity weaker than that of the benchmark antibody, serulizumab.

[0035] Figure 6 Shows that the anti-Claudin 18.2 / CD40 bispecific antibody binds to human B cells with an activity weaker than that of the benchmark antibody, serulizumab.

[0036] Figure 7 Shows that the CD40 monospecific antibody activates CD40 signaling with an activity far lower than that of the benchmark antibody, serulizumab.

[0037] Figure 8 Shows that the anti-Claudin 18.2 / CD40 bispecific antibody activates CD40 signaling in a Claudin 18.2-dependent manner.

[0038] Figure 9 Shows that the anti-Claudin 18.2 / CD40 bispecific antibody activates dendritic cells to secrete IL-12 in a Claudin 18.2-dependent manner.

[0039] Figure 10 Shows that 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 Shows that the anti-Claudin 18.2 / CD40 bispecific antibody increases B cell proliferation (A) and activation (B) in a Claudin 18.2-dependent manner.

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

[0042] Figure 13 Shows that the humanized anti-Claudin 18.2 / CD40 bispecific antibody has CD40 activation activity comparable to that of its chimeric antibody.

[0043] Figure 14 Shows that the humanized anti-Claudin 18.2 / CD40 bispecific antibody has a function of inducing IL-12 secretion comparable to that of its chimeric antibody.

[0044] Figure 15 It is shown that the humanized anti-Claudin 18.2 / CD40 bispecific antibody has the function of inducing the expression of CD80 (A) and CD86 (B) on dendritic cells, which is comparable to that of its chimeric antibody.

[0045] Figure 16 It is shown that the humanized anti-Claudin 18.2 / CD40 bispecific antibody has the function of inducing B cell proliferation (A) and activation (B), which is comparable to that of its chimeric antibody.

[0046] Figure 17 The in vivo tumor growth inhibition of the anti-Claudin 18.2 / CD40 chimeric bispecific antibody is shown.

[0047] Figure 18 The in vivo study design (A), blood biochemical analysis (B), tumor-infiltrating immune phenotype (IPT) analysis (C-F), and spleen IPT analysis (G-I) of the anti-Claudin 18.2 / CD40 chimeric bispecific antibody are shown.

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

[0049] Figure 20 It is shown that the anti-5T4 / CD40 bispecific antibody activates dendritic cells to secrete IL-12 in a 5T4-dependent manner.

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

[0051] Figure 22 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 are shown.

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

[0053] Figure 24 It is shown that anti-5T4 / CD40 bispecific antibodies with different formats activate CD40 signaling with different potencies.

[0054] Figure 25Show that anti-5T4 / CD40 bispecific antibodies with different formats activate dendritic cells to secrete IL-12 with different potencies.

[0055] Figure 26 Show the in vivo tumor growth inhibition of anti-5T4 / CD40 bispecific antibodies.

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

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

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

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

[0060] Figure 31 Show that the humanized anti-5T4 / CD40 bispecific antibody b16(42p155z2)-LALA has a function of inducing IL-12 secretion comparable to its chimeric antibody. Detailed Description

[0061] Definitions

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

[0063] A polynucleotide or polynucleotide region (or polypeptide or polypeptide region) having a certain percentage (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) of "sequence identity" with another sequence means that when aligned, the percentage of bases (or amino acids) is the same when comparing the two sequences. Such alignment and the percentage of homology or sequence identity can be determined using software programs known in the art, such as those described in Ausubel et al. eds. (2007) "Current Protocols in Molecular Biology". 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; expect = 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 specified percentage of homology as described above 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 with a certain degree of homology or sequence identity with the nucleotide sequence of the nucleic acid or its complement. Homologs of double-stranded nucleic acids are intended to include nucleic acids having a nucleotide sequence with a certain degree of homology with it or its complement. In one aspect, the homolog of a nucleic acid is capable of hybridizing to the nucleic acid or its complement. Similarly, an "equivalent polypeptide" refers to a polypeptide having 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 to the reference polypeptide or polynucleotide, the equivalent polypeptide or polynucleotide has one, two, three, four or five additions, deletions, substitutions and combinations thereof. In some aspects, the equivalent sequence retains the activity (e.g., epitope binding) or structure (e.g., salt bridge) of the 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 full antibody and any antigen-binding fragment or single chain thereof. Thus, the term "antibody" includes any protein or peptide containing a molecule that comprises at least a portion of an immunoglobulin molecule having biological activity of binding to an antigen. Examples thereof include, but are not limited to, complementarity-determining regions (CDRs) of a heavy or light chain or ligand-binding portions thereof, variable regions of a heavy or light chain, constant regions of a heavy or light chain, framework (FR) regions, or any portion thereof, or at least a portion of a binding protein.

[0066] Single-domain antibodies (sdAbs), also known as nanobodies, are antibody fragments consisting of a single monomeric variable antibody domain. Nanobodies produced by camelids and certain other animals are also known as VHH fragments. Like full antibodies, nanobodies are capable of selectively binding to a specific antigen. Due to a molecular weight of only 12 kDa to 15 kDa, single-domain antibodies are much smaller than ordinary antibodies (150 kDa to 160 kDa). Single-domain antibodies, due to their small size and single-chain nature, can be particularly suitable as fragments incorporated in other proteins such as chimeric antigen receptors (CARs) and bispecific antibodies.

[0067] As used herein, the term "antibody fragment" or "antigen-binding fragment" is a part 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 a full antibody. The term "antibody fragment" includes aptamers, mirror-image isomers, and diabodies. The term "antibody fragment" also includes any synthetic or genetically engineered protein that acts in a manner similar to an antibody by binding to a specific antigen to form a complex.

[0068] The antibodies, antigen-binding polypeptides, variants or derivatives thereof of the present disclosure 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 containing VK or VH domains, fragments produced by a Fab expression library, and anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies against the LIGHT antibodies disclosed herein). The immunoglobulin or antibody molecules of the present disclosure 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] "Specifically binds" or "is specific for" generally means that an antibody binds to an epitope via its antigen-binding domain, and this binding requires some complementarity between the antigen-binding domain and the epitope. By this definition, an antibody is considered to "specifically bind" an epitope when it binds the epitope via its antigen-binding domain more readily than it binds a random, unrelated epitope. The term "specificity" is used herein to define the relative affinity of a given antibody for a given epitope. For example, antibody "A" may be considered to have a higher specificity for a given epitope than antibody "B", or antibody "A" may be considered to bind 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 prophylactic or defensive measures, wherein the goal is to prevent or slow down (alleviate) an undesired physiological change or condition, such as cancer progression. Beneficial or desired clinical outcomes include, but are not limited to, alleviation of detectable or undetectable symptoms, diminishment of the extent of a disease, stabilization (i.e., not worsening) of a disease state, delay or slowing of disease progression, improvement or palliation of a disease state, and remission (partial or complete). "Treatment" can also refer to an extended survival as compared to the expected survival of an individual not receiving treatment. Individuals in need of treatment include those already suffering from a condition or disorder as well as those susceptible to a condition or disorder or those in which a condition or disorder is to be prevented.

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

[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 present disclosure for, for example, detection, diagnostic procedures, and / or treatment.

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

[0074] CD40 is widely expressed in hematopoietic and non-hematopoietic tissues. CD40 regulates immunity and thus 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 independent of innate immune receptors such as stimulator of interferon genes (STING) or Toll-like receptors (TLRs). Accordingly, a great deal 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 with respect to dosing and scheduling, 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. Current solutions are to treat these toxicities when they occur, which is difficult to manage, costly, and off-putting to patients.

[0076] Through careful design and screening, unexpectedly, the inventors were able to identify CD40 agonist antibodies with different CD40 activation activities in the presence or absence of tumor-associated antigen (TAA). In particular, a long list of new CD40 antibodies was identified, all of which had significantly reduced CD40 agonist activity compared to the reference antibody, sirukumab (the most widely studied CD40 antibody candidate) (see, for example, Example 4 and Figure 8 A). However, when used in a bispecific or multispecific format that also contains an anti-TAA moiety, these antibodies exhibit far greater CD40 activation activity than sirukumab (see, for example, Figure 8 B, Figures 9 to 11 ).

[0077] These antibodies can thus be termed "TAA-dependent CD40 agonist antibodies". These TAA-dependent CD40 agonist antibodies have shown greatly enhanced anti-tumor efficacy in animal models (see, for example, Example 9 and Figure 16 ). It is also inevitable that they will result in greatly reduced toxicity, as 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 are further classified into four classes. As Figure 8 shown and summarized in Table 5, class 4 antibodies do not activate CD40 in the absence of TAA and have only relatively weak CD40 activation in the presence of TAA; class 3 antibodies have moderate CD40 activation in the absence of TAA and the most potent CD40 activation in the presence of TAA; and class 2 antibodies have marginal CD40 activation in the absence of TAA and moderate CD40 activation in the presence of TAA.

[0079] Class 1 antibodies are of the most interest. They have low levels of CD40 activation or no CD40 activation in the absence of TAA, and effective CD40 activation when TAA is present on target cells. Class 1 antibodies include 42p155, 2p834, 2p931, 42p655, and 2p1294. These antibodies are thought to have the highest therapeutic index and thus the greatest clinical potential.

[0080] Accordingly, 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 moiety, activates CD40 only on cells expressing the TAA and activates more than CD40 on reference cells lacking the TAA. For a fair comparison, in some embodiments, the reference cells differ from the cells expressing the TAA only in the expression of the TAA.

[0081] In some embodiments, the difference in CD40 activation between the cells expressing the TAA and the 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 activation of CD40 by the TAA-dependent CD40 agonist antibody in the absence of the TAA is lower than that of serulimumab, such as 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% lower than serulimumab.

[0083] In some embodiments, the TAA on the cells expressing the TAA 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 the tumors expressing the TAA.

[0084] In some embodiments, the activation measurement is 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] Various assays for measuring CD40 activation are available, including using 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 by IL-12 secretion, CD80, and CD86 expression. In yet another example, the target cells are B cells and the CD40 activation is measured by Ki67 and / or CD86 expression.

[0086] Exemplary TAA-dependent CD40 agonist antibodies are also provided that are as long as proteins (such as 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, 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 have the CDR1, CDR2, and CDR3 sequences of antibody 42p155 (SEQ ID NO:1), respectively. In some embodiments, the CDR1, CDR2, and CDR3 comprise the amino acid sequences of SEQ ID NO:14-16, respectively.

[0087] Analysis shows that certain residues in CDR2 of 42p155 may undergo post-translational modification (PTM). Therefore, mutations are made to prevent such PTM (hence called PTM de-risked forms), including NG => NA or QG. See, for example, SEQ ID NO:63 and 64. Thus, in some embodiments, the CDR1, CDR2, and CDR3 comprise the amino acid sequences of SEQ ID NO:14, 63, and 16, respectively. Thus, in some embodiments, the CDR1, CDR2, and CDR3 comprise the amino acid sequences of SEQ ID NO:14, 64, and 16, respectively.

[0088] In some embodiments, humanized forms of 42p155 and their PTM de-risked counterparts, such as those provided in SEQ ID NO:53-62, are also provided. In some embodiments, the humanized antibody comprises back mutations selected from 1P, 2S, 88P, and 98Q according to Kabat numbering. In some embodiments, the humanized antibody comprises the back mutation 98Q. In some embodiments, the humanized antibody comprises the back mutations 88P and 98Q. In some embodiments, the humanized antibody comprises the back mutations 1P, 2S, 88P, and 98Q.

[0089] In some embodiments, in the 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 NO: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 NO:53, 54, 57, or 60.

[0090] In some embodiments, in the 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 NO: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 NO:55, 58, or 61.

[0091] In some embodiments, in the 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 NO: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 NO: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 have the CDR1, CDR2, and CDR3 sequences of antibody 2p834 (SEQ ID NO:2), respectively. In some embodiments, the CDR1, CDR2, and CDR3 comprise the amino acid sequences of SEQ ID NO:17-19, respectively.

[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 back mutations selected from 1P, 2S, 88P, and 98Q according to Kabat numbering. In some embodiments, the humanized antibody comprises the back mutation 98Q. In some embodiments, the humanized antibody comprises the back mutations 88P and 98Q. In some embodiments, the humanized antibody comprises the back mutations 1P, 2S, 88P, and 98Q. 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 with SEQ ID NO: 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 have the CDR1, CDR2, and CDR3 sequences of antibody 2p931 (SEQ ID NO: 3), respectively. In some embodiments, the CDR1, CDR2, and CDR3 comprise the amino acid sequences of SEQ ID NOs: 20 - 22, respectively. 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 with 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 have the CDR1, CDR2, and CDR3 sequences of antibody 42p655 (SEQ ID NO: 4), respectively. In some embodiments, the CDR1, CDR2, and CDR3 comprise the amino acid sequences of SEQ ID NOs: 23 - 25, respectively. 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 with 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 have the CDR1, CDR2, and CDR3 sequences of antibody 2p1294 (SEQ ID NO:5), respectively. In some embodiments, the CDR1, CDR2, and CDR3 comprise the amino acid sequences of SEQ ID NOs: 26-28, respectively. 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 with 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 have the CDR1, CDR2, and CDR3 sequences of antibody 2p957 (SEQ ID NO:6), respectively. In some embodiments, the CDR1, CDR2, and CDR3 comprise the amino acid sequences of SEQ ID NOs: 29-31, respectively. 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 with 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 have the CDR1, CDR2, and CDR3 sequences of antibody 42p495 (SEQ ID NO:7), respectively. In some embodiments, the CDR1, CDR2, and CDR3 comprise the amino acid sequences of SEQ ID NOs: 32-34, respectively. 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 with 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 have the CDR1, CDR2, and CDR3 sequences of antibody 3p78 (SEQ ID NO:8), respectively. In some embodiments, the CDR1, CDR2, and CDR3 comprise the amino acid sequences of SEQ ID NOs: 35-37, respectively. 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 with 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 have the CDR1, CDR2, and CDR3 sequences of antibody 2p415 (SEQ ID NO:9), respectively. In some embodiments, the CDR1, CDR2, and CDR3 comprise the amino acid sequences of SEQ ID NOs: 38-40, respectively. 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 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 have the CDR1, CDR2, and CDR3 sequences of antibody 2p442 (SEQ ID NO:10), respectively. In some embodiments, the CDR1, CDR2, and CDR3 comprise the amino acid sequences of SEQ ID NOs: 41-43, respectively. 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 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 antibody comprises the recited CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: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 have the CDR1, CDR2, and CDR3 sequences of antibody 2p551 (SEQ ID NO:11), respectively. In some embodiments, the CDR1, CDR2, and CDR3 comprise the amino acid sequences of SEQ ID NOs: 44-46, respectively. 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 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 have the CDR1, CDR2, and CDR3 sequences of antibody 2p80 (SEQ ID NO: 12), respectively. In some embodiments, the CDR1, CDR2, and CDR3 comprise the amino acid sequences of SEQ ID NOs: 47-49, respectively. 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 with 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 have the CDR1, CDR2, and CDR3 sequences of antibody 2p1130 (SEQ ID NO: 13), respectively. In some embodiments, the CDR1, CDR2, and CDR3 comprise the amino acid sequences of SEQ ID NOs: 50-52, respectively. 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 with SEQ ID NO: 14.

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

[0107] Compositions comprising the antibody or the polypeptide and a pharmaceutically acceptable carrier are also provided.

[0108] Those of ordinary skill in the art will also understand that the antibodies disclosed herein can be modified such that they differ in amino acid sequence from the naturally occurring binding polypeptides from which they are derived. For example, the polypeptide or amino acid sequence derived from a designated protein can be similar, e.g., having a certain percentage identity with the starting sequence, e.g., it can be 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to the starting sequence. In some embodiments, the modified antibody or fragment retains the designated CDR sequences.

[0109] In certain embodiments, the antibody comprises an amino acid sequence or one or more moieties that are not normally associated with an antibody. Exemplary modifications are described in more detail below. For example, the antibodies of the present disclosure may comprise a flexible linker sequence or may be modified to add a functional moiety (e.g., PEG, drug, toxin, or label).

[0110] Bispecific and multispecific antibodies

[0111] As provided, the CD40 agonist antibodies disclosed herein are particularly useful for preparing bispecific and multispecific antibodies. This is at least because of their enhanced therapeutic index and their small size.

[0112] CD40 is widely expressed in hematopoietic and non-hematopoietic tissues. CD40 regulates immunity and thus 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 independent of innate immune receptors such as stimulator of interferon genes (STING) or Toll-like receptors (TLRs). Accordingly, a great deal of work has been done to develop CD40 agonist antibodies for the treatment of cancer.

[0113] However, unlike inhibitory antibodies, the agonist approach presents significant challenges with respect to dosing and scheduling, which complicates drug development. Another complicating factor is that CD40 agonist antibodies are generally 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 when they occur, which is difficult to manage, costly, and off-putting to patients.

[0114] Accordingly, in one embodiment, there is provided a bispecific antibody comprising a TAA-dependent CD40 agonist antibody or an antigen-binding fragment thereof, and a second antibody or antigen-binding fragment having binding specificity for a target antigen other than CD40. In some embodiments, a third or fourth specificity is also included. The target antigen other than CD40 is a tumor antigen in some embodiments.

[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 moiety, activates CD40 on cells expressing the TAA and activates more than CD40 on reference cells lacking the TAA. For fair comparison, in some embodiments, the reference cells differ from the cells expressing the TAA only in the expression of the TAA.

[0116] In some embodiments, the difference in CD40 activation between cells expressing a 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 in the absence of TAA than zalutumumab, such as less than zalutumumab by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%.

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

[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 using 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 foregoing section, such as 42p155, 2p834, 2p931, 42p655, 2p1294, 2p957, 42p495, 3p78, 2p415, 2p442, 2p551, 2p80, and 2p1130 and their biological equivalents.

[0122] The abundance of tumor antigens is known in the art, and new tumor antigens can be readily 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 inventors designed a panel of 5T4 x CD40 bispecific antibodies that have anti-5T4 / anti-CD40 moieties with different properties and are in different formats. These bispecific antibodies were evaluated in CD40 reporter cells and co-cultured with target cells expressing 5T4. Potency 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. In addition, in vivo anti-tumor efficacy was determined in CD40 humanized C57BL / 6 mice bearing MC38-hu5T4 tumors.

[0124] Bispecific antibodies that activate CD40 signaling in a 5T4-dependent manner have been observed to display optimal in vitro and in vivo performance. In addition, bispecific antibodies in multiple formats were tested, and two of them (b16 and b18) showed superiority in inducing more effective CD40 agonism in a 5T4-dependent manner.

[0125] In vivo testing with 5T4 x CD40 demonstrated effective anti-tumor efficacy, which was significantly higher than the clinical benchmark at similar doses. In addition, the resulting tumor-free mice were resistant to tumor rechallenge, demonstrating the establishment of a durable memory response. In addition, ex vivo analysis showed focused immune activation in the tumor without peripheral activation, ensuring the safety of these bispecific antibodies.

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

[0127] According to one embodiment of the present disclosure, there is provided a bispecific antibody or a multispecific antibody incorporating the bispecific antibody, which comprises an anti-5T4 moiety and an anti-CD40 moiety. In some embodiments, the anti-CD40 moiety 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 which have significantly reduced CD40 agonist activity compared to the reference antibody, sirukumab, the most widely studied CD40 antibody candidate. When used in a bispecific or multispecific format which also contains units of an anti-tumor associated antigen (TAA, such as 5T4), these antibodies exhibit far greater CD40 activation activity than sirukumab. These antibodies can thus be referred to as "5T4-dependent CD40 agonist antibodies". These 5T4-dependent CD40 agonist antibodies have shown greatly enhanced anti-tumor efficacy in animal models. It is also inevitable that they will result in greatly reduced toxicity as they do not induce CD40 activity in tissues or organs which do not express 5T4.

[0129] A 5T4-dependent CD40 agonist antibody is an antibody which, when presented in a bispecific or multispecific antibody format which also contains anti-5T4 units, activates CD40 only on cells which express 5T4 and activates more CD40 on reference cells which lack 5T4. For 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 5T4-expressing cells 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 in the absence of 5T4 than sirukumab, such as less than sirukumab by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%.

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

[0133] In some embodiments, the activation measurement is 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] Various assays for measuring CD40 activation are available, including using 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] Exemplary 5T4-dependent CD40 agonist antibodies are also provided, which are as long as proteins (such as bispecific 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, 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 have the CDR1, CDR2, and CDR3 sequences of antibody 42p155 (SEQ ID NO:1), respectively. In some embodiments, the CDR1, CDR2, and CDR3 comprise the amino acid sequences of SEQ ID NOs: 14-16, respectively.

[0136] Analysis shows that certain residues in the CDR2 of 42p155 may undergo post-translational modification (PTM). Therefore, mutations are made to prevent such PTM (thus called PTM de-risked forms), including NG => NA or QG. See, for example, SEQ ID NOs: 63 and 64. Therefore, in some embodiments, the CDR1, CDR2, and CDR3 comprise the amino acid sequences of SEQ ID NOs: 14, 63, and 16, respectively. Therefore, 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-de-risked counterparts, such as those provided in SEQ ID NOs: 53-62, are also provided. In some embodiments, the humanized antibody includes back mutations selected from 1P, 2S, 88P, and 98Q according to Kabat numbering. In some embodiments, the humanized antibody includes the back mutation 98Q. In some embodiments, the humanized antibody includes the back mutations 88P and 98Q. In some embodiments, the humanized antibody includes the back mutations 1P, 2S, 88P, and 98Q.

[0138] In some embodiments, in the 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 the 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 the 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 thereof are also provided that compete with any antibody of the present disclosure for binding to human CD40. In some embodiments, anti-CD40 antibodies and antigen-binding fragments thereof are also provided that bind to the same epitope as any antibody of the present disclosure. In some embodiments, anti-CD40 antibodies and antigen-binding fragments thereof are also provided that comprise CDR1, CDR2, and CDR3 of the antibodies of the present disclosure.

[0142] One of ordinary skill in the art will also understand that the antibodies disclosed herein can be modified such that they differ in amino acid sequence from the naturally occurring binding polypeptides from which they are derived. For example, the polypeptide or amino acid sequence derived from a designated protein can be similar, e.g., having a certain percentage identity with the starting sequence, e.g., it can be 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to the starting sequence. In some embodiments, the modified antibody or fragment retains the designated CDR sequences.

[0143] B. Bispecific antibodies with anti-5T4 moieties targeting Bin A or B

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

[0145] As provided in Example 10, all of the anti-5T4 antibodies disclosed herein can be classified into four segments, A - D, according to a binding competition assay. Segment A includes the 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, the antibodies of segments B and C exhibit excellent agonist activity.

[0146] The anti-5T4 antibodies and antigen-binding fragments of segment A can be represented as antibody 14G12, its humanized and de-risked forms, and those that compete with 14G12 for binding to the human 5T4 protein.

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

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

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

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

[0151] The anti-5T4 antibodies and antigen-binding fragments of Region A may also be represented as antibody 393E9, its humanized and de-risked forms, 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 that comprises a heavy-chain variable region (VH) and a light-chain variable region (VL), the heavy-chain variable region comprising CDR1, CDR2, and CDR3, and the light-chain variable region comprising CDR1, CDR2, and CDR3. In some embodiments, the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 comprise the sequences of SEQ ID NOs: 103-108, respectively.

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

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

[0155] The anti-5T4 antibodies and antigen-binding fragments of Region B may be represented as antibody 159D5, its humanized and de-risked forms, 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 thereof, which comprises a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region comprising CDR1, CDR2, and CDR3, and the light chain variable region comprising CDR1, CDR2, and CDR3. In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 comprise the sequences of SEQ ID NOs: 123-128, respectively.

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

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

[0159] In certain embodiments, for certain desired activities, the anti-5T4 moiety may include an antibody or antigen-binding fragment of domain D, which may be represented as antibody 286B4, its humanized and de-risked forms, 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 thereof that comprises a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region comprising CDR1, CDR2, and CDR3, and the light chain variable region comprising CDR1, CDR2, and CDR3. In some embodiments, the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 comprise the sequences of SEQ ID NOs: 140-145, respectively.

[0161] In some embodiments, VH and VL comprise the sequences of SEQ ID NOs: 138 and 139, respectively. In some embodiments, VH comprises any one of the sequences of SEQ ID NOs: 146-151, and VL comprises any one of the sequences of SEQ ID NOs: 152-157. In some embodiments, VH comprises the recited 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 VL comprises the recited 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] Multiple bispecific antibody formats have been tested in the appended examples. Format b11 ( Figure 4 and Figure 22 A) exhibited excellent activity characteristics, while formats b16 ( Figure 22 D) and b18 ( Figure 22 F) showed 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 prevent binding to FcγR. Exemplary mutations include leucine (L) to alanine (A) substitutions at positions 234 and 235 (LALA) and alanine (A) to asparagine (N) substitution at position 297 (N297A).

[0164] In format b16, one of the VH / VL pairs in a conventional Fab 5T4 antibody is replaced by two tandem anti-CD40 VHH antibodies. This 1+2 format is thus a heterodimer. The Fc portion may optionally be mutated to abrogate ADCC / CDC activity or prevent binding to FcγR. Exemplary mutations include leucine (L) to alanine (A) substitutions (LALA) at positions 234 and 235 and alanine (A) to asparagine (N) substitution (N297A) at position 297. Additionally, the Fc fragment may 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 3 rather than just 2 VHH anti-CD40 antibodies. Again, the Fc portion may optionally be mutated to abrogate ADCC / CDC activity or prevent binding to FcγR. Exemplary mutations include leucine (L) to alanine (A) substitutions (LALA) at positions 234 and 235 and alanine (A) to asparagine (N) substitution (N297A) at position 297. Additionally, the Fc fragment may be mutated to incorporate a knob-in-hole to reduce mispairing.

[0166] According to one embodiment of the present disclosure, there is thus provided a bispecific antibody or a multispecific antibody incorporating the bispecific antibody, which comprises a 5T4-binding portion and a CD40-binding portion. In some embodiments, the 5T4-binding portion comprises a conventional heavy chain-light chain pair. In some embodiments, the CD40-binding portion comprises at least two serially fused VHH antibodies. In some embodiments, the CD40-binding portion comprises at least three serially fused VHH antibodies. In some embodiments, the 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 abrogate ADCC / CDC activity or prevent its binding to FcγR. Exemplary mutations include leucine (L) to alanine (A) substitutions (LALA) at positions 234 and 235 and alanine (A) to asparagine (N) substitution (N297A) at position 297. Additionally, the Fc fragment may be mutated to incorporate a knob-in-hole to reduce mispairing.

[0168] In some embodiments, bispecific antibodies or multispecific antibodies incorporating the bispecific antibodies are provided, which include an anti-5T4 moiety and an anti-CD40 moiety. In some embodiments, the anti-5T4 moiety comprises a conventional Fab antibody. In some embodiments, the anti-CD40 moiety comprises two separate VHH antibodies, each of which is fused to the C-terminus of one of the two chains of the 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 abolish ADCC / CDC activity or prevent its binding to FcγR. Exemplary mutations include leucine (L) to alanine (A) substitutions (LALA) at positions 234 and 235 and alanine (A) to asparagine (N) substitution (N297A) at position 297. Additionally, the Fc fragment can be mutated to incorporate a knob-in-hole to reduce mismatching.

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

[0171] Chimeric antigen receptor (CAR)

[0172] Chimeric antigen receptors (CARs) comprising the nanobodies of the present disclosure are also provided. In the CAR, the nanobody can serve as the antigen recognition domain. Additionally, in some embodiments, the CAR further comprises an extracellular hinge region, a transmembrane domain, and an intracellular T cell signaling domain.

[0173] The hinge, also known as the spacer, is a small domain located between the antigen recognition region and the cell outer membrane. A suitable hinge enhances the flexibility of the scFv receptor head and reduces the steric hindrance between the CAR and its target antigen. Exemplary hinge sequences are based on the juxtamembrane regions of immunological molecules such as IgG, CD8, and CD28.

[0174] The transmembrane domain is a structural component consisting of a hydrophobic α-helix spanning the cell membrane. It anchors the CAR to the plasma membrane, bridging the extracellular hinge and antigen recognition domain with the intracellular signaling region. Generally, transmembrane domains from the juxtamembrane components of intracellular domains, such as the CD28 transmembrane domain, can be used.

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

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

[0177] Polynucleotides encoding antibodies and methods of making antibodies

[0178] The present disclosure also provides isolated polynucleotides or nucleic acid molecules encoding the antibodies, variants, or derivatives of the present disclosure. The polynucleotides of the present disclosure can encode the entire heavy and light chain variable regions of the antigen-binding polypeptide, its variants, or derivatives on the same polynucleotide molecule or on separate polynucleotide molecules. Additionally, the polynucleotides of the present disclosure can encode portions of the heavy and light chain variable regions of the antigen-binding polypeptide, its variants, or derivatives 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 target cells to express the antibody or a fragment thereof.

[0180] The mRNA can be synthesized according to any of a variety of known methods. For example, the mRNA can be synthesized by in vitro transcription (IVT). Briefly, IVT generally involves a linear or circular DNA template containing a promoter, a pool of ribonucleoside triphosphates, a buffer system that may contain DTT and magnesium ions, and an appropriate RNA polymerase (e.g., T3, T7, or SP6 RNA polymerase), DNase I, pyrophosphatase, and / or 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 the desired nucleotide sequence for the mRNA encoding the desired antibody (e.g., encoding a heavy or light chain) and a termination signal.

[0182] The mRNA sequence encoding the desired antibody (e.g., encoding the heavy or light chain) can be determined and incorporated into a DNA template using standard methods. For example, starting from the desired amino acid sequence (e.g., the desired heavy or light chain sequence), virtual reverse translation is performed based on the degenerate genetic code. An optimization algorithm can then be used to select suitable codons. Generally, on the one hand, the G / C content can be optimized to achieve the highest possible G / C content, and on the other hand, the frequency of tRNA can be considered as much as possible according to codon usage. The optimized RNA sequence can be established and displayed, for example, with the aid of a suitable display device, and compared with the original (wild-type) sequence. The secondary structure can also be analyzed to calculate the stabilizing and destabilizing properties or regions of the RNA, respectively.

[0183] mRNA can be synthesized as unmodified or modified mRNA. Generally, mRNA is modified to enhance stability. Modifications of mRNA can include, for example, modifications of RNA nucleotides. Modified mRNA can thus include, for example, backbone modifications, sugar modifications, or base modifications. In some embodiments, mRNA encoding an antibody (e.g., mRNA encoding a heavy chain and a light chain) can 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)), and modified nucleotide analogs or derivatives of purines and pyrimidines, such as, for example, 1-methyl-adenine, 2-methyl-adenine, 2-methylthio-N6-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, pseudouridine (5-uridine), dihydro-uridine, 2-thio-uridine, 4-thio-uridine, 5-carboxymethylaminomethyl-2-thio-uridine, 5-(carboxyhydroxymethyl)-uridine, 5-fluoro-uridine, 5-bromo-uridine, 5-carboxymethylaminomethyl-uridine, 5-methyl-2-thio-uridine, 5-methyl-uridine, N-uridine-5-oxyacetic acid methyl ester, 5-methylaminomethyl-uridine, 5-methoxyaminomethyl-2-thio-uridine, 5'-methoxycarbonylmethyl-uridine, 5-methoxy-uridine, uridine-5-oxyacetic acid methyl ester, uridine-5-oxyacetic acid (v), 1-methyl-pseudouridine, queosine, 13-D-mannosyl-queosine, wybutoxosine, and phosphoramidates, thiophosphates, 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 herein by reference in their entireties.

[0184] In some embodiments, the mRNA (e.g., mRNA encoding a heavy chain and a light chain) may contain RNA backbone modifications. Generally, backbone modifications are modifications in which the phosphate of the nucleotide backbone contained in the RNA is chemically modified. Exemplary backbone modifications typically include, but are not limited to, modifications from methylphosphonate, methylaminophosphate, aminophosphate, phosphorothioate (e.g., cytidine 5'-O-(1-thiophosphate)), boranophosphate, positively charged guanidinium groups, etc., which means replacing the phosphodiester bond with other anionic, cationic, or neutral groups.

[0185] In some embodiments, the mRNA (e.g., mRNA encoding a heavy chain and a light chain) may contain sugar modifications. Typical sugar modifications are chemical modifications of the sugar of the nucleotide, which include, but are not limited to, sugar modifications selected from 2'-deoxy-2'-fluoro-oligoribonucleotides (2'-fluoro-2'-deoxycytidine 5'-triphosphate, 2'-fluoro-2'-deoxyuridine 5'-triphosphate), 2'-deoxy-2'-deamino-oligoribonucleotides (2'-amino-2'-deoxycytidine 5'-triphosphate, 2'-amino-2'-deoxyuridine 5'-triphosphate), 2'-O-alkyl oligoribonucleotides, 2'-deoxy-2'-C-alkyl oligoribonucleotides (2'-O-methylcytidine 5'-triphosphate, 2'-methyluridine 5'-triphosphate), 2'-C-alkyl oligoribonucleotides and their isomers (2'-aracytidine 5'-triphosphate, 2'-arouridine 5'-triphosphate), or azidotriphosphates (2'-azido-2'-deoxycytidine 5'-triphosphate, 2'-azido-2'-deoxyuridine 5'-triphosphate).

[0186] In some embodiments, the mRNA (e.g., mRNA encoding a heavy chain and a light chain) can contain modifications of the bases of the 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 ribonucleoside 5'-triphosphate, 2-aminoadenosine 5'-triphosphate, 2-thiocytidine 5'-triphosphate, 2-thiouridine 5'-triphosphate, 4-thiouridine 5'-triphosphate, 5-aminoallyl cytidine 5'-triphosphate, 5-aminoallyl uridine 5'-triphosphate, 5-bromocytidine 5'-triphosphate, 5-bromouridine 5'-triphosphate, 5-iodocytidine 5'-triphosphate, 5-iodouridine 5'-triphosphate, 5-methylcytidine 5'-triphosphate, 5-methyluridine 5'-triphosphate, 6-azacytidine 5'-triphosphate, 6-azauridine 5'-triphosphate, 6-chloropurine ribonucleoside 5'-triphosphate, 7-deazaadenosine 5'-triphosphate, 7-deazaguanosine 5'-triphosphate, 8-azaaadenosine 5'-triphosphate, 8-azidoadenosine 5'-triphosphate, benzimidazole ribonucleoside 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 includes adding 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" is used to protect the mRNA from exonucleolytic degradation.

[0188] Thus, in some embodiments, the mRNA (e.g., mRNA encoding a heavy chain and a light chain) contains a 5' cap structure. The 5' cap is typically added as follows: First, an RNA terminal phosphatase removes a terminal phosphate group from the 5' nucleotide, leaving two terminal phosphates; then guanosine triphosphate (GTP) is added to the terminal phosphate via guanylyltransferase, generating a 5'5'5 triphosphate bond; then the 7-nitrogen of guanine is methylated with 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, the mRNA (e.g., mRNA encoding a heavy chain and a light chain) comprises a 3' poly(A) tail structure. The poly(A) tail on the 3' end of the mRNA typically comprises from about 10 to 300 adenosine nucleotides (e.g., from about 10 to 200 adenosine nucleotides, from about 10 to 175 adenosine nucleotides, from about 10 to 150 adenosine nucleotides, from about 10 to 125 adenosine nucleotides, 10 to 100 adenosine nucleotides, from about 10 to 75 adenosine nucleotides, from about 20 to 70 adenosine nucleotides, or from about 20 to 60 adenosine nucleotides). In some embodiments, the mRNA encoding an antibody (e.g., mRNA encoding a heavy chain and a light chain) comprises a 3' poly(C) tail structure. A suitable poly(C) tail on the 3' end of the mRNA typically comprises from about 10 to 200 cytosine nucleotides (e.g., from about 10 to 150 cytosine nucleotides, from about 10 to 100 cytosine nucleotides, from about 20 to 70 cytosine nucleotides, from about 20 to 60 cytosine nucleotides, or from 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, the mRNA (e.g., mRNA encoding a heavy chain and a light chain) comprises a 5' and / or 3' untranslated region. In some embodiments, the 5' untranslated region comprises one or more elements that affect mRNA stability or translation, such as an iron response element. In some embodiments, the 5' untranslated region can be from about 50 to 500 nucleotides in length (e.g., from about 50 to 400 nucleotides in length, from about 50 to 300 nucleotides in length, from about 50 to 200 nucleotides in length, or from about 50 to 100 nucleotides in length).

[0191] In some embodiments, the 5' region of the mRNA (e.g., mRNA encoding a heavy chain and a light chain) comprises a sequence encoding a signal peptide, such as those described herein. In certain embodiments, a signal peptide derived from human growth hormone (hGH) is incorporated into the 5' region. Typically, the signal peptide coding sequence is directly or indirectly linked to the heavy chain 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 against a desired antigen 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, both 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, by administering an antigen to a transgenic animal that has been modified to produce such antibodies in response to antigen challenge, but whose endogenous loci have been disabled, fully human antibodies specific for a particular antigen can be prepared. Exemplary techniques useful for preparing such antibodies are described in U.S. Pat. Nos. 6,150,584, 6,458,592, 6,420,140, which are hereby incorporated by reference in their entirety.

[0194] In certain embodiments, the antibodies prepared do not elicit a harmful immune response in the animal to be treated (e.g., in humans). In one embodiment, the antigen-binding polypeptides, variants or derivatives of the present disclosure are modified using techniques well recognized in the art to reduce their immunogenicity. For example, the 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 parental antibody but have reduced immunogenicity in humans. This can be achieved by various methods, including (a) grafting an entire non-human variable domain onto a human constant region to produce a chimeric antibody; (b) grafting at least a portion of one or more non-human complementarity determining regions (CDRs) into a human framework and constant region that retains or does not retain critical framework residues; or (c) grafting an entire non-human variable domain but "masking" them with human-like segments by substituting 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. Immunol. 25:489-498 (1991); Padlan, Molec. Immunol. 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 herein.

[0195] Deimmunization can also be used to reduce the immunogenicity of antibodies. As used herein, the term "deimmunization" includes modifying an antibody to modify T cell epitopes (see, e.g., International Application Publication Nos.: WO / 9852976A1 and WO / 0034317A2). For example, the variable heavy chain and variable light chain sequences from a starting antibody are analyzed, and a human T cell epitope "map" is generated from each V region, which shows the positions of epitopes relative to the complementarity determining regions (CDRs) and other key residues within the sequence. Individual T cell epitopes from the T cell epitope map are analyzed in order to identify alternative amino acid substitutions that have a low risk of altering the final antibody activity. A series of alternative variable heavy chain sequences and variable light chain sequences are designed, the design including combinatorial amino acid substitutions, and these sequences are subsequently incorporated into a series of binding polypeptides. Typically, 12 to 24 variant antibodies are generated and tested for their binding and / or function. The full-length heavy and light chain genes containing the modified variable regions and human constant regions are then cloned into an expression vector, and the plasmid is subsequently introduced into a cell line to produce the full antibody. The antibodies are then compared in appropriate biochemical and biological assays, and the best 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, particularly cold tumors

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

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

[0200] The antibodies of the present disclosure can also be used to treat or inhibit cancer. In some embodiments, a tumor antigen (e.g., Claudin 18.2) is overexpressed in tumor cells. Thus, in some embodiments, a method for treating cancer in a patient in need thereof is provided. The method in one embodiment requires administering to the patient an effective amount of an antibody of the present disclosure. 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. Inducing gene expression can be carried out, 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, kidney cancer, and small cell lung cancer. Accordingly, the antibodies of the present disclosure can be used to treat any one or more of such cancers.

[0202] In some embodiments, the tumors being treated are those that are particularly challenging to treat with conventional immuno-oncology therapies, such as antibodies targeting immune checkpoints (ICPs). Sometimes, such tumors are referred to as "cold tumors" or "non-immunogenic tumors". CD40 activation can convert the so-called cold tumors (non-immunogenic tumors) into hot tumors. In some embodiments, accordingly, the present disclosure 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 defective in T cell filtration, antigen-presenting cells (APCs), or T cell activation, or 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. Additionally, 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] The identification of non-immunogenic tumors 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)) describe a standardized scoring system, Immunoscore, which is based on the quantification of two lymphocyte populations (CD4 and CD8) (e.g., in resected tissue) to guide the differentiation between hot and cold tumors. The Immunoscore ranges from Immunoscore 0 (I0, low density, such as the lack of both cell types in two regions) to I4 (high density of immune cells in two locations). By classifying cancers based on their immune infiltration, the scoring system provides an immune-based tumor classification, including definitions 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 immune checkpoint inhibitors such as PD-L1 inhibitors, PD-1 inhibitors, CTLA-4 inhibitors, or combinations 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, a patient treated with a CD40 agonist antibody (or multispecific antibody) of the present disclosure is further treated with a second anti-cancer agent. In some embodiments, the second anti-cancer agent is an immune checkpoint inhibitor, such as an antibody specific for PD-1, PD-L1, or CTLA-4, but is not limited thereto. In some embodiments, the second anti-cancer agent is administered together with the CD40 agonist antibody (or multispecific antibody) of the present disclosure. In some embodiments, the second anti-cancer agent is administered before or after administration of the CD40 agonist antibody (or multispecific antibody) of the present disclosure.

[0207] Other conditions or disorders associated with increased cell survival that can be treated, prevented, diagnosed, and / or prognosed using the antibodies or variants or derivatives of the present disclosure include, but are not limited to, the progression and / or metastasis of malignancies and associated disorders, such as leukemia (including acute leukemia (e.g., acute lymphocytic leukemia, acute myeloid leukemia (including myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, and erythroleukemia)) and chronic leukemia (e.g., chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphoma (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, osteosarcoma, chordoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's 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, cholangiocarcinoma, 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 being 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 particular disease being treated. The judgment of healthcare providers regarding such factors is within the ordinary skill in the art. The amount will also depend on the individual patient to be treated, the route of administration, the type of formulation, the properties of the compound being 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 methods of administration of the antibodies, variants, or compositions include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The antigen-binding polypeptide or composition can be administered by any convenient route, such as by infusion or bolus injection, absorbed through an epithelial or mucocutaneous lining (such as oral mucosa, rectal, and intestinal mucosa, etc.), and can be administered together with other bioactive agents. Thus, the pharmaceutical composition containing the antigen-binding polypeptide of the present disclosure can be administered orally, rectally, parenterally, intracisternally, vaginally, intraperitoneally, topically (such as by powder, ointment, drops, or transdermal patch), sublingually, or as an oral or nasal spray.

[0210] As used herein, the term "parenteral" refers to a mode of administration that includes intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous, and intra-articular injections and infusions.

[0211] The administration can 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 injections; intraventricular injection can be facilitated by an intraventricular catheter, for example, connecting the catheter to a reservoir, such as an Ommaya reservoir. Pulmonary administration can also be employed, such as by using an inhaler or nebulizer and a nebulized formulation.

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

[0213] Composition

[0214] The present disclosure also provides pharmaceutical compositions. Such compositions contain 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 state government or listed in the U.S. Pharmacopeia or other recognized pharmacopeias for use in animals, and more specifically in humans. In addition, a "pharmaceutically acceptable carrier" is generally any type of non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation aid.

[0216] The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a therapeutic agent is administered. Such pharmaceutical carriers can be sterile liquids such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. When a pharmaceutical composition is administered intravenously, water is a preferred carrier. Aqueous solutions of saline as well as glucose and glycerol are also 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, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, ethylene glycol, water, ethanol, etc. If desired, the composition may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, such as acetates, citrates or phosphates. Antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; and tonicity regulators such as sodium chloride or glucose may also be considered. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, etc. Compositions may be formulated into suppositories using conventional binders and carriers such as triglycerides. Oral formulations may contain standard carriers such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, saccharin sodium, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences by E.W. Martin, which is incorporated herein by reference. Such compositions will contain a therapeutically effective amount of the antigen-binding polypeptide, preferably in purified form, and a suitable amount of carrier in order to provide a form suitable for proper administration to a patient. The formulation should be suitable for the mode of administration. The parent formulation may be encapsulated in an ampoule, disposable syringe or multi-dose vial made of glass or plastic.

[0217] In one embodiment, the composition is formulated, according to conventional procedures, into a pharmaceutical composition suitable for intravenous administration to humans. Generally, compositions for intravenous administration are solutions in sterile isotonic aqueous buffers. When necessary, the composition may also contain solubilizing agents and local anesthetics (such as lidocaine) to relieve the pain at the injection site. Generally, these components are provided individually or mixed together in unit dosage forms, for example, as a lyophilized powder or an anhydrous concentrate in a sealed container (such as an ampoule or sachet) indicating the amount of the active agent. In the case where the composition is administered by infusion, it may be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. In the case where the composition is administered by injection, a vial of sterile water for injection or saline may be provided in order to mix the components before administration.

[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, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2 - aminoethanol, histidine, procaine, etc.

[0219] Example

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

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

[0222] Immunization: To generate VHH antibodies against human CD40, two alpacas were immunized with human CD40 protein. After 4 rounds of immunization, the antibody titers of the immunized alpaca sera were evaluated 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 the VHH fragment in the 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 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 an antigen for phage library panning.

[0225] Phage library solution panning against human CD40: The bound phages were eluted with Gly - Hcl. The resulting phages were Output 1. The bound phages were incubated with SS320 cells and plated on 2YT plates for the next round of panning screening. A total of 3 rounds of panning screening were performed. After three rounds of screening, phage ELISA of Output 1, Output 2, and Output 3 showed enriched CD40 binders.

[0226] Single clones were selected from Output 2 and Output 3 phages. Antigen - binding ELISA was performed on the phages of these clones. 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. ELISA binding evaluation of the purified antibodies was performed on 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 2p1130 are listed in Tables 1 and 1A through 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. CDR Sequences of 2p931

[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 1E. CDR Sequences of 2p1294

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

[0242] Table 1F. CDR Sequences of 2p957

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

[0244] Table 1G. CDR Sequences of 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. CDR Sequences of 2p415

[0249]

[0250]

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

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

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

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

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

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

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

[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] The binding activity of the antibody to CD40 protein was tested in this example.

[0261] 2.1 ELISA binding to CD40

[0262] To evaluate the binding activities of clones 42p155, 2p834, 2p931, 42p655, 2p1294, 2p957, 42p495, 3p78, 2p415, 2p442, 2p551, 2p80 and 2p1130, ELISA tests were performed on chimeric monospecific antibodies (mAbs) from these clones and serulimab (i.e., the clinical benchmark anti-CD40 agonist antibody).

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

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

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

[0266] <![CDATA[Clone / 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] --: No binding

[0268] 2.2 Binding to cell surface CD40

[0269] To evaluate their binding affinities to cell surface CD40, chimeric anti-CD40 monospecific antibodies were tested in the Jurkat cell line overexpressing CD40 by FACS. In FACS buffer, a total of 1×10 5 Jurkat-CD40 cells per well were incubated with 10-fold serial dilutions of antibodies starting from 100 nM for 30 min at 4 °C. After washing with FACS buffer, conjugated PE anti-human IgG antibody was added to each well and incubated for 30 min at 4 °C. After washing, the MFI of PE was evaluated by MACSQuant Analyzer 16. As Figure 2 shown, the antibodies tested showed concentration-dependent binding ability to CD40.

[0270] 2.3 Protein full kinetics of CD40

[0271] Using the capture method, the binding of antibodies to the anti-Claudin-18.2 moiety against recombinant CD40 protein (human CD40-his tag) was tested by Biacore, and these antibodies were in monospecific antibody format (cAb) or bispecific format (BiAb, as Figure 4(as shown). Use a Protein A chip to capture bispecific antibodies with each of the 42p155, 2p834, 2p931, 42p655, 2p1294, 2p957, 42p495, 3p78, 2p415, 2p442, 2p551, and 2p1130 clones and the 2p80 monospecific antibody. Inject serially diluted human CD40-His-tagged protein onto the captured antibodies at a flow rate of 30 μl / min for 2 minutes. Dissociate the antigen 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 different binding / dissociation patterns.

[0272] Table 4: Full kinetics measured by Biacore

[0273]

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

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

[0276] Briefly, microtiter plates were coated overnight at 4 °C with 1 μg / ml human 4-1BB protein or human OX40 protein in PBS at 100 μl / well, and then blocked with 1% BSA at 150 μl / well. Ten-fold dilutions of the 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. As Figure 3 shown, none of these clones showed cross-reactivity with 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, a bispecific antibody (BiAb) containing an anti-CD40 nanobody and an anti-claudin 18.2 (CLDN18.2) unit was generated and tested. Two anti-CD40 fragments of 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 in a 2+2 bispecific antibody format (see the description in 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 the bispecific antibody to human CD40 on human dendritic cells

[0280] In the presence of IL-4 and GM-CSF, human DCs were induced from human CD14+ cells for 6 to 7 days. The above 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 an FcR blocking reagent (MACS) at 4°C for 15 minutes, and then incubated with 10-fold serial dilutions of the claudin 18.2 / CD40 bispecific antibody starting from 100 nM at 4°C for 30 minutes. A 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 a MACSQuant Analyzer 16.

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

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

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

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

[0285] Example 4. Functional activity of CD40 nanobody

[0286] In this example, the functional activity of the antibody was tested and it was shown that, unlike serulizumab, the VHH chimeric antibody activated CD40 signaling only at a low level.

[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 the 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 was induced when the antibody bound to the CD40 receptor. Briefly, reporter cells at a density of 2.5×10 4 cells / well were cultured in white 96-well plates. The antibodies were serially diluted 10-fold and added to the white 96-well assay plates 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 the luciferase substrate and measured by a microplate reader. Four-parameter logistic curve analysis was performed using GraphPad software.

[0289] As Figure 7As shown, serulimab monoclonal antibody dose-dependently activates CD40 signaling. Antibodies 42p155, 2p834, 2p931, 42p655, 2p1294, 2p957, 42p495, 3p78, 2p415, 2p442, 2p551, 2p80, and 2p1130 induced CD40 signaling only at doses higher than 10 nM, and in the same experimental setup, their maximum RLU values were all less than half of the highest value of serulimab.

[0290] Example 5. Functional Activity of Anti-Claudin 18.2 / CD40 Bispecific Antibody

[0291] In this example, the activity of the anti-Claudin 18.2 / CD40 bispecific antibody generated in the Figure 4 shown format was tested in a CD40 NF-κB luciferase reporter system, as well as the activity of promoting human dendritic cell immune response and B cell immune response.

[0292] 5.1 Cell Line-Based Functional Characterization of 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 gene system was used. In this assay, H_CD40 (TNFRSF5) NFκB-reporter Jurkat (Genomeditech, cat# GM-C09520) was used as effector cells, and cells expressing CHO-K1 or not expressing Claudin 18.2 were used as target cells. Briefly, effector cells at a density of 2.0×10 4 cells / well were co-cultured with 2.0×10 4 target cells (E / T ratio = 1:1) in white 96-well plates. The antibody was serially diluted 10-fold and added to the white 96-well assay plates 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 the luciferase substrate and measured using a microplate reader. Four-parameter logistic curve analysis was performed using GraphPad software.

[0294] As Figure 8As shown, serulimab monoclonal antibody can dose-dependently enhance CD40 signal transduction in cells overexpressing CHO-K1 and CHO-claudin-18.2. 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 the cells and exhibit much stronger activation of CD40 signal transduction in the presence of claudin-18.2. In addition, the bispecific antibodies show diverse CD40 activities. Based on EC 50 and the highest value, the CD40 clones were divided into four categories (levels 1 to 4), as shown in Table 5.

[0295] Table 5. Potency categories

[0296]

[0297] 5.2 Activity of bispecific antibodies in promoting immune responses of human dendritic cells

[0298] To study the ability of the claudin-18.2-CD40 bispecific antibody to stimulate the response of human dendritic cells (DCs), the IL-12 cytokine release and CD80 / CD86 expression of DCs were detected.

[0299] Human DCs were obtained according to the procedure specified 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 cells) were co-cultured with CHO-K1-claudin-18.2 or parental CHO-K1 cells (1.5×10 4 cells) (E / T ratio = approximately 3:1). The bispecific antibody was serially diluted 10-fold and added to the medium at a final concentration starting from 100 nM. After incubation for 48 hours, the IL-12 level in the medium was measured using the IL-12 / p40 (human) LANCE Ultra TR-FRET detection kit (PerkinElmer). The data were analyzed using non-linear regression, i.e., the four-parameter logistic equation.

[0300] Activation of DCs leads to upregulation of co-stimulatory molecules CD80 / 86. Here, according to the staining and analysis procedures, the CD80 / CD86 expression of DCs was detected by FACS. Briefly, the 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 a MACSQuant Analyzer 16.

[0301] As Figure 9 and Figure 10 shown, serulizumab monoclonal antibody can dose-dependently activate the DC response in cells overexpressing CHO-K1 and CHO-claudin 18.2 ([[]] Figure 9 IL-12 secretion in [[[]] Figure 10 and CD80 / CD86 expression in A-B). The bispecific antibody can only activate the DC response in the presence of cells overexpressing claudin 18.2. And the potency is related to the expression level of CLDN18.2

[0302] 5.3 Activity of bispecific antibody in promoting human B cell immune response

[0303] To study 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. According to the manufacturer's protocol, human B cells were isolated from human PBMCs using a B cell isolation kit. 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 cells) were co-cultured with CHO-K1-claudin 18.2 or parental CHO-K1 cells (1×10[[[]] 4 cells) (E / T ratio = 5:1). The bispecific antibody was serially diluted 10-fold and added to the medium at a final concentration starting from 100 nM. After incubation for 72 hours, according to the staining and analysis procedures, the Ki67 and CD86 expression of B cells were detected by FACS. 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 the Foxp3 / Transcription Factor Staining Buffer Set (Invitrogen), and then Alexa The 488 anti-human Ki-67 antibody was stained for 30 minutes. After washing, the MFIs of AF488 and BV421 gated on CD19+ cells were evaluated by a MACSQuant analyzer 16.

[0304] As Figure 11 shown, the serulimab monoclonal antibody can dose-dependently activate the B cell response in cells overexpressing CHO-K1 and CHO-claudin 18.2 ( Figure 11 Ki67 / CD86 expression in A / B), while the bispecific antibody can only activate the B cell response in the presence of cells overexpressing claudin 18.2 and the potency is related to 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 42p155 and 2p442 variable region genes. In the first step of this method, the amino acid sequences of the VHHs of 42p155 and 2p442 were compared with the available human Ig gene sequence database to find the overall best-matching human germline Ig gene sequences. Then, the humanized variable domain sequences of 42p155 and 2p442 were designed, where CDRH1, H2, and H3 were located on the framework sequences of their VH genes respectively.

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

[0308] Table 6-1. Humanized 42p155 antibody sequence (underlined indicates CDR; bold / italic indicates back mutation; bold indicates PTM removal)

[0309]

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

[0311]

[0312]

[0313] Table 6-2. Humanized 2p442 antibody sequence (underlined indicates CDR; bold / italic indicates back mutation)

[0314]

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

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

[0317] The humanized VHH gene was 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 Overall Kinetic Affinities 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 the capture method, respectively.

[0321] For the humanized 42p155 monoclonal antibody, 42p155z2 and 42p155z3 were captured using a Protein A chip. Serial dilutions of human CD40-His tag protein were injected onto the captured antibody at a flow rate of 10 μl / min for 3 minutes. The antigen was allowed to dissociate for 6 minutes. All experiments were performed on a Biacore T200. Data analysis was performed using the 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 tag protein were incubated with the captured antibody for 5 minutes. The antigen was allowed to dissociate for 10 minutes. All experiments were performed on an Octet RED96e. Data analysis was performed using Octet Analysis Studio 12.2 software.

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

[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 the humanized 2p442 monoclonal antibody to cell surface CD40, 2p442z8, 2p442z9, 2p442z10 and parental 2p442 were tested in the Jurkat cell line overexpressing CD40 by FACS. In FACS buffer, a total of 1×10 5 Jurkat-CD40 cells per well were incubated with antibodies serially diluted 3-fold starting from 100 nM at 4 °C for 30 minutes. After washing with FACS buffer, anti-human IgG antibody conjugated with PE was added to each well and incubated at 4 °C for 30 minutes. After washing, the MFI of PE was evaluated by MACSQuant Analyzer 16. As Figure 12 shown, the humanized 2p442 antibody showed binding activity comparable to that of the parental chimeric antibody.

[0330] Example 8. Functional Activity of Humanized Bispecific Antibodies

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

[0332] Table 8. Humanized Antibody Sequences (Underlined Indicates CDR; Bold / Italic Indicates Back Mutations)

[0333]

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

[0335] To evaluate the ability of the humanized Claudin 18.2-CD40 bispecific antibody 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 Claudin 18.2 were used as target cells. In a white 96-well plate, the effector cells and target cells were co-cultured at an E / T ratio of 1:1. The antibody was serially diluted 5-fold and added to the white 96-well assay plate at a final concentration range of 0.001 nM to 100 nM. Luminescence was obtained after incubation with the luciferase substrate for 5 hours and measured using a microplate reader.

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

[0337] 8.2 Activity of humanized bispecific antibody in promoting human dendritic cell immune response

[0338] To study the ability of the humanized CLDN18.2 / CD40 bispecific antibody to stimulate the response of human dendritic cells (DCs), the release of the IL-12 cytokine and the expression of CD80 / CD86 of DCs were detected as described in Example 5 above. Human DCs were obtained according to the procedure specified 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 cells) were co-cultured with CHO-K1-Claudin 18.2 or parental CHO-K1 cells (1.5×10 4 cells) (E / T ratio = approximately 3:1). The humanized bispecific antibody was added to the medium at a final concentration starting from 100 nM. After incubation for 48 hours, the IL-12 level in the medium was measured using the IL-12 / p40 (human) LANCEUltra TR-FRET detection kit (PerkinElmer). In addition, according to the staining and analysis procedures described in Example 5.2, the expression of CD80 / CD86 of DCs was detected by FACS. As Figure 14 and Figure 15 shown, the DC responses induced by the humanized bispecific antibody ( Figure 14 IL-12 secretion in Figure 15 and CD80 / CD86 expression in

[0339] 8.3 Activity of humanized bispecific antibody in promoting human B cell immune response

[0340] To study the ability of a humanized Claudin 18.2-CD40 bispecific antibody to activate human B cells, the Ki67 (cell proliferation) and CD86 expression of B cells were detected. 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 cells) were co-cultured with CHO-K1-Claudin 18.2 or parental CHO-K1 cells (1×10 4 cells) (E / T ratio = 5:1). Five-fold serially diluted humanized bispecific antibodies were added to the medium at final concentrations 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. As Figure 16 shown, the B cell responses induced by humanized bispecific antibodies ( Figure 16 Ki67 / CD86 expression in A / B) were comparable to those of 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 murine CD40 was replaced with the corresponding human CD40 were used to test the anti-tumor activity of the bispecific antibody.

[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). As Figure 17 shown in A, the following antibodies were administered intraperitoneally to the mice twice a week for a total of 6 times: human IgG control (3.6 mg / kg), cerulizumab (3 mg / kg), 42p155 anti-CLDN18.2 / CD40 bispecific antibody (3.6 mg / kg, equimolar to cerulizumab), 2p834 anti-CLDN18.2 / CD40 bispecific antibody (3.6 mg / kg, equimolar to cerulizumab).

[0344] As Figure 17 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 cerulizumab only showed a moderate response. In addition, 51 days after the first treatment, MC38-hCLDND18.2 cells were subcutaneously injected again into the contralateral flanks of the tumor-free mice (at Figure 17which are represented by black arrows in B) in order to determine the formation of immune cell memory against the tumor cell line. As a control, 4 naive C57bl / 6-huCD40 mice were injected with the same MC38-hCLDND18.2 tumor cells. During the experiment, tumor volume was monitored twice a week by caliper measurement.

[0345] As Figure 17 shown in B and Table 9, serulizumab was able to inhibit tumor growth with a TGI of 78.3% at day 23 after the first treatment. Meanwhile, the 42p155 and 2p834 bispecific antibodies induced complete tumor remission in all treated mice starting from day 27. In addition, after re-challenge, there was no MC38-hCLDND18.2 tumor growth in the group previously treated with the 42p155 and 2p834 bispecific antibodies, while there was 100% tumor growth in naive mice. These results indicate that the anti-tumor efficacy and tumor growth regression of the anti-CD40 therapy are stronger compared to non-targeted CD40 therapies. The re-challenge 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 Serulizumab 78.3％ 42p155BiAb 103.2％ 2p834BiAb 102.8％

[0348] To evaluate the immune activation of the anti-CD40 therapy, tumor infiltrating immunophenotyping (IPT) analysis was performed on day 7 after intraperitoneal administration, twice a week for a total of 2 times ( Figure 18 A). As Figure 18 shown in C-F, the 42p155 bispecific antibody elicited a significant immune response in tumor tissues, including an increase in the percentages of leukocytes, T cells (CD8 + and CD4 + T cells), DCs, and B cells compared to the PBS and serulizumab groups. In contrast, serulizumab only slightly increased the percentage of T cells, especially CD8 + T cells. In addition, compared to the PBS and serulizumab groups, the 42p155-BiAb also enhanced the proliferation of CD8 T cells, CD4 T cells, and B cells, as well as increased the expression of CD80 and CD86 in DCs and B cells. These data indicate that the 42p155 bispecific antibody can significantly enhance the immune response in tumor tissues, superior to the anti-CD40 agonistic monoclonal antibody.

[0349] To determine whether the anti-CD40 therapy has any effect on the peripheral immune system, immunophenotyping (IPT) analysis of the spleen was also performed on day 7. AsFigure 18 As shown in G-I, 42p155-BiAb did not significantly affect peripheral immune cell populations, while serulizumab reduced T cells. At the same time, activated B cells also increased after serulizumab treatment. These data indicate that serulizumab activates peripheral CD40, while 42p155-BiAb remains silent in the periphery.

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

[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 mice, Balb / C mice, and C57BL / 6 mice were first immunized with 5T4-His protein. Subsequently, the immunized mice were boosted with 5T4-His protein or human 5T4 expressing CHO-K1. To select mice that produce antibodies binding to the 5T4 protein, the antibody titers of the sera of the immunized mice were evaluated by ELISA and FACS. Briefly, microtiter plates were coated overnight at 4°C with 0.5 μg / mL or 1 μg / mL of human 5T4 or cynomolgus monkey 5T4 protein in ELISA coating buffer at 100 μL / well, and then blocked with 1% BSA at 150 μL / well. Dilutions of sera from the 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 by spectrophotometer at OD 450 nm. The immune response against the CHOK1-hu5T4 cell line was also tested by serum FACS, where the CHOK1 parental cell line was used as a negative control. The resulting mice were used for fusion. Hybridoma supernatants were screened by ELISA.

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

[0356] Screening: Hybridoma supernatants were screened for recombinant human (rh) 5T4-His and recombinant cynomolgus monkey 5T4-His proteins by ELISA. Then, the positive supernatants from the primary screening were confirmed by FACS binding to the CHOK1-hu5T4 cell line and by ELISA binding to the 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. The subclones were screened in the same manner as the primary clones, and the culture supernatants of the positive clones were further confirmed by affinity ranking.

[0358] Clones 14G12, 393E9, 159D5, and 286B4 were selected for further analysis and humanization. By competitive ELISA, the tested 5T4 mAbs were classified into four segments (Segment A, Segment B, Segment C, and Segment D) based on the binding epitopes on human 5T4. As a reference, the Fab portion of natupromab 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 used to generate humanized mAbs. In the first step of this method, the amino acid sequences of VH and VK were compared with the available human Ig gene sequence databases to find the overall best-matching human germline Ig gene sequences.

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

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

[0363]

[0364]

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

[0366]

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

[0368]

[0369]

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

[0371]

[0372]

[0373] Table 10-5. Humanization of 159D5 (Underlining Indicates CDRs; Bold / Italic Indicates Back Mutations)

[0374]

[0375]

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

[0377]

[0378] Table 10-7. Humanization of 286B4 (Underlining Indicates CDRs; Bold / Italic Indicates Back Mutations, Boxes Indicate Potential PTM Site Removal)

[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 tested the functional activity of the 5T4-CD40 bispecific antibody and selected the 5T4 portion of the 5T4-CD40 bispecific antibody.

[0385] Generation of 5T4-CD40 Bispecific Antibodies with Different 5T4 Binding Epitopes

[0386] As Figure 4As shown, the 5T4 sequences of 14G12, 393E9, 159D5, and 286B4 were constructed into the 5T4 portion in the "2 + 2b11" format. 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 portion. The peptide chains of the "2 + 2b11" format bispecific antibody are shown in Tables 11A - D.

[0387] Table 11A. Peptide Chains of 14G12 - 42p155 Bispecific Antibody

[0388]

[0389] Table 11B. Peptide Chains of 393E9 - 42p155 Bispecific Antibody

[0390]

[0391]

[0392] Table 11C. Peptide Chains of 159D5 - 42p155 Bispecific Antibody

[0393]

[0394] Table 11D. Peptide Chains of 286B4 - 42p155 Bispecific Antibody

[0395]

[0396] Cell - Line - Based Functional Characterization of 5T4 - CD40 Bispecific Antibodies with Different 5T4 Binding Epitopes

[0397] To evaluate the ability of 5T4 - CD40 bispecific antibodies to activate the CD40 signaling pathway, a commercial CD40NF - 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 cells not expressing it (CHO - K1) were used as target cells. Briefly, effector cells at a density of 2.0×10 4 cells / well were combined with 2.0×10 4Target cells (E / T ratio = 1:1) were co-cultured in white 96-well plates. The antibody was serially diluted 10-fold and added to the white 96-well assay plates at final concentrations ranging from 0.001 nM to 100 nM. After incubation at 37 °C for 5 hours, luminescence was obtained by adding luciferase substrate and measured using a microplate reader. Four-parameter logistic curve analysis was performed using GraphPad software.

[0398] As Figure 19 shown, the activities of 5T4-CD40 bispecific antibodies with different epitopes in the 5T4 protein were only dependent 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, 5T4-CD40 bispecific antibodies with segments A and B containing the 5T4 portion showed strong agonist activity, especially when the target cells expressed moderate levels of hu5T4 on the surface.

[0399] Activity of 5T4-CD40 bispecific antibody in promoting human dendritic cell immune response

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

[0401] Human DCs were obtained according to the procedure specified in Example 4. Human DCs were used as effector cells. Cells expressing human 5T4 (MCF-7:5T4 低 and CHO-K1-hu5T4:5T4 高 ) or cells not expressing it (CHO-K1) were used as target cells. Human DCs (5 × 10 4 cells) were co-cultured with target cells (1.5 × 10 4 ) (E / T ratio = approximately 3:1). The bispecific antibody was serially diluted 10-fold and added to the medium at final concentrations starting from 100 nM. After incubation for 48 hours, the IL-12 level in the medium was measured using the IL-12 / p40 (human) LANCE Ultra TR-FRET detection kit (PerkinElmer). The data were analyzed using non-linear regression, namely the 4-parameter logistic equation.

[0402] Activation of DCs leads to upregulation of the costimulatory molecules CD80 / CD86. Here, the CD80 / CD86 expression of DCs was detected by FACS according to the staining and analysis procedures. Briefly, the stimulated DCs were harvested by pipetting and washed with FACS buffer. After blocking with FcR blocking reagent (MACS) for 15 minutes at 4°C, 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 a MACSQuant Analyzer 16.

[0403] As Figure 20 and Figure 21 shown, the 5T4-CD40 bispecific antibody can activate the DC response only in the presence of cells expressing hu5T4. And the potency is related to the expression level of hu5T4. Similarly, the 5T4-CD40 bispecific antibodies with 5T4 moieties in segment A and segment B showed strong agonist activity.

[0404] Example 13. Generation of Bispecific Antibodies with Different Bispecific Antibody Formats

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

[0406] Table 12A. Peptide Chains of Exemplary Bispecific Antibodies

[0407]

[0408]

[0409]

[0410]

[0411]

[0412]

[0413]

[0414]

[0415]

[0416]

[0417]

[0418]

[0419]

[0420] Table 12B. Antibody variable region sequences of the CD40 moiety

[0421]

[0422]

[0423] Table 12C. Antibody variable region sequences of the 5T4 moiety for use in / suitable for bispecific antibodies

[0424]

[0425] Example 14. Selection of 5T4 - CD40 formats with different CD40 activities

[0426] To select a suitable format for the 5T4 - CD40 bispecific antibody, we tested the functional activities of 5T4 - CD40 bispecific antibodies with different formats and CD40 activities to select the most effective and cleanest combination of the 5T4 - CD40 bispecific antibody.

[0427] Binding activity of the bispecific antibody to human CD40 on the cell surface

[0428] To evaluate the binding affinity of the 5T4 - CD40 bispecific antibody to cell - surface CD40, bispecific antibodies with b11(42p155z2), b12(42p155z2), b13(42p155z2), b16(42p155z2), b17(42p155z2), and b18(2p1130) formats were tested by FACS in the Jurkat cell line overexpressing CD40. In FACS buffer, 1×10 5 Jurkat - CD40 cells per well were incubated with 5 - fold serial dilutions of the antibody starting from 100 nM at 4°C for 30 minutes. After washing with FACS buffer, a PE - conjugated anti - human IgG antibody was added to each well and incubated at 4°C for 30 minutes. After washing, the MFI of PE was evaluated by a MACSQuant Analyzer 16. As Figure 23 shown, the binding activities of the tested 5T4 - CD40 bispecific antibodies were dependent on the antibody format as well as the CD40 clone. In addition, asFigure 29 As shown, the humanized form of 5T4-CD40 biAb, namely b16(42p155z2)-LALA, showed comparable binding to the chimeric antibody b16(42p155z2) against CD40 expression on DCs.

[0429] Functional characterization of 5T4-CD40 bispecific antibodies with different formats and CD40 clones based on cell lines

[0430] To evaluate the potency of 5T4-CD40 bispecific antibodies in activating the CD40 signaling pathway, a commercial CD40NF-κB luciferase reporter gene system was used. In this assay, H_CD40(TNFRSF5)NFκB-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 cells not expressing it (CHO-K1) were used as target cells. Briefly, effector cells at a density of 2.0×10 4 cells / well were co-cultured with 2.0×10 4 target cells (E / T ratio = 1:1) in white 96-well plates. The antibodies were serially diluted 10-fold and added to the white 96-well assay plates 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 the luciferase substrate and measured by a microplate reader. Four-parameter logistic curve analysis was performed using GraphPad software.

[0431] As Figure 24 shown, the activities of the tested 5T4-CD40 bispecific antibodies with different formats and CD40 clones were only dependent 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 other formats. In addition, as Figure 30 shown, when 5T4 was expressed, the humanized form of 5T4-CD40 biAb, namely b16(42p155z2)-LALA, showed comparable activity to its chimeric antibody b16(42p155z2) in inducing CD40 downstream NF-κB signaling.

[0432] Activity of 5T4-CD40 bispecific antibodies in promoting human dendritic cell immune responses

[0433] To study the ability of 5T4-CD40 bispecific antibodies to stimulate human dendritic cell (DC) responses, the release of the cytokine IL-12 from DCs was measured.

[0434] Human DCs were obtained according to the procedure specified in Example 4. Human DCs were used as effector cells. Cells expressing human 5T4 (MCF-7:5T4 低 and CHO-K1-hu5T4:5T4 高 ) or cells not expressing it (CHO-K1) were used as target cells. Human DCs (5×10 4 cells) were co-cultured with target cells (1.5×10 4 ) (E / T ratio = approximately 3:1). The bispecific antibodies were serially diluted 10-fold and added to the medium at final concentrations starting from 100 nM. After incubation for 48 hours, the IL-12 level in the medium was measured using the IL-12 / p40 (human) LANCE Ultra TR-FRET detection kit (PerkinElmer). The data were analyzed using non-linear regression, namely the 4-parameter logistic equation.

[0435] As Figure 25 shown, the activities of the 5T4-CD40 bispecific antibodies tested with different formats and CD40 clones were only dependent 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. Similarly, the potencies of the 5T4-CD40 bispecific antibodies tested were significantly different, and the bispecific antibodies with b16 and b18 formats showed much stronger agonist activity than other antibodies. In addition, as Figure 31 shown, when 5T4 was expressed, the humanized form of 5T4-CD40 biAb, namely b16(42p155z2)-LALA, showed comparable activity to its chimeric antibody b16(42p155z2) in inducing DCs to secrete IL2.

[0436] Example 15. Inhibition of tumor growth by 5T4-CD40 bispecific antibodies

[0437] In this example, humanized mice in which the extracellular domain of murine CD40 was replaced with the human counterpart were used to test the anti-tumor activity of 5T4-CD40 bispecific antibodies.

[0438] Murine colon adenocarcinoma cells (MC38) were engineered to express human 5T4 (MC38-hu5T4). MC38-hu5T4 cells were implanted subcutaneously into humanized C57BL / 6 mice (huCD40). As Figure 26As shown in A, the following antibodies were administered intraperitoneally to mice twice a week for a total of 4 times: PBS control, serulizumab (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 the tested antibodies were administered in equimolar amounts.

[0439] As Figure 26 shown in B and Table 13, mice treated with serulizumab and b16(2p1130) showed only a moderate response. On day 25, serulizumab inhibited tumor growth with a TGI of 76.1%, and b16(2p1130) inhibited tumor growth with a TGI of 83.8%. Meanwhile, b18(2p1130) (3 mg / kg) and b16(42p155z2) induced complete tumor remission in 4 / 6 and 5 / 6 mice, respectively, starting from day 39. And all mice treated with b18(2p1130) (10 mg / kg) had complete tumor regression starting from day 35. These results indicate that compared with non-targeted CD40 therapy, 5T4-targeted CD40 therapy has strong anti-tumor efficacy and tumor growth regression. To determine whether CD40-targeted therapy can induce immune memory, a second challenge of MC38-hu5T4 was given to the contralateral flank of mice with complete tumor remission. As Figure 26 shown in C, all these mice were resistant to tumor rechallenge, indicating the establishment of a durable immune memory response in mice treated with the 5T4-CD40 bispecific antibodies b18(2p1130) and b16(42p155z2).

[0440] Immunophenotyping (IPT) analysis in peripheral blood was performed on day 7 after the first administration. As Figure 27 shown, compared with the PBS group, b16(2p1130) and b16(42p155z2) did not change the cell count of B cells, while serulizumab and b18(2p1130) decreased the cell number ( Figure 27 A). In addition, serulizumab also significantly increased the expression of CD80 and CD86 in B cells and the proliferation of T cells, while the other bispecific antibodies did not change or only slightly increased the activation markers ( Figure 27 B - C). These data indicate that b16(2p1130) and b16(42p155z2) did not activate immune cells in the periphery.

[0441] To evaluate the immune activation of CD40-based therapy, tumor-infiltrating IPT analysis was performed on day 7 in a separate group with the same experimental design ( Figure 28 A). As Figure 28As shown in B-E, compared with the PBS group, b16(42p155z2) induced an immune response in tumor tissues, including an increase in the percentages of leukocytes, CD4+ T cells, and DCs. In addition, compared with the PBS group, b16(42p155z2) also increased the expression of CD80 and CD86 in DCs. These data indicate that the 5T4-CD40 bispecific antibody is a promising therapeutic approach for treating 5T4-expressing tumors through 5T4-dependent CD40 activation.

[0442] Table 13. TGI of the MC38-hu5T4 mouse model

[0443] Group TGI(%) of D25 Serulizumab (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％

[0444] ***

[0445] The scope of the present disclosure is not limited by the specific embodiments described, which are intended to be illustrative of individual aspects of the present disclosure, and any compositions or methods that are functionally equivalent are within the scope of the present disclosure. It will be apparent to those skilled in the art that various modifications and variations can be made to the methods and compositions of the present disclosure without departing from the spirit or scope of the present disclosure. Accordingly, the present disclosure is intended to cover various modifications and variations of the present disclosure, provided that they fall within the scope of the appended claims or their equivalents.

[0446] All publications and patent applications mentioned in this specification are incorporated herein by reference to the extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Claims

1. A single-domain antibody, wherein the single-domain antibody has binding specificity for human cluster of differentiation 40 (CD40) protein and comprises complementarity-determining region 1 (CDR1), CDR2, and CDR3, wherein the CDR1, CDR2, and CDR3 are respectively: (1) The amino acid sequences of SEQ ID NO: 20, 21, and 22; (2) The amino acid sequences of SEQ ID NO: 50, 51, and 52; or (3) The amino acid sequences of SEQ ID NO: 17, 18, and 19.

2. The antibody according to claim 1, wherein the CDR1 is the amino acid sequence of SEQ ID NO: 20, the CDR2 is the amino acid sequence of SEQ ID NO: 21, and the CDR3 is the amino acid sequence of SEQ ID NO:

22.

3. The antibody according to claim 2, wherein the antibody comprises the amino acid sequence of SEQ ID NO:

3.

4. The antibody according to claim 1, wherein the CDR1 is the amino acid sequence of SEQ ID NO: 50, the CDR2 is the amino acid sequence of SEQ ID NO: 51, and the CDR3 is the amino acid sequence of SEQ ID NO:

52.

5. The antibody according to claim 4, wherein the antibody comprises the amino acid sequence of SEQ ID NO:

13.

6. The antibody according to claim 1, wherein the CDR1 is the amino acid sequence of SEQ ID NO: 17, the CDR2 is the amino acid sequence of SEQ ID NO: 18, and the CDR3 is the amino acid sequence of SEQ ID NO:

19.

7. The antibody according to claim 6, wherein the antibody comprises the amino acid sequence of SEQ ID NO:

2.

8. One or more polynucleotides encoding the antibody according to any one of claims 1 to 7.

9. The polynucleotide according to claim 8, wherein the polynucleotide is one or more mRNAs; wherein the mRNA is chemically modified.

10. A cell comprising the polynucleotide according to claim 9.

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