Humanized anti-human βig-h3 protein and its uses

Abstract

The development of tumor stroma during cancer plays a key role as it may act as a physical barrier and restrict the access of immune cells to the tumor. Thus, overexpression of βig-h3 (TGFβi) in the stroma is associated with poor prognosis in pancreatic ductal adenocarcinoma and other cancers. A monoclonal antibody against βig-h3 protein, called 18B3, has been shown to play a role in directly altering antitumor immune responses by blocking the inhibition of CD8+ T cell activation. The inventors have developed humanized antibodies from 18B3 with unexpectedly high affinity, slow dissociation rates, and strong thermal stability. These humanized antibodies are therefore strong candidates for treating cancers in which the stroma expresses βig-h3 in vivo. The present invention thus relates to these humanized monoclonal antibodies and methods for treating such cancers.

Description

[Technical field]

[0001] The present invention relates to the field of antibodies. In particular, the present invention provides a humanized antibody with specificity for human βig-h3 protein and its uses. Medical uses are also provided, particularly for the treatment of cancers in which the stromal protein βig-h3 is expressed in vivo, such as pancreatic ductal adenocarcinoma (PDAC), lung cancer, head and neck cancer, colorectal cancer, bladder cancer, melanoma, and other cancers as described below. [Background technology]

[0002] The development of tumor stroma during cancer plays a key role as it may act as a physical barrier restricting the access of immune cells to the tumor. Therefore, the identification of key molecules expressed or overexpressed in the tumor stroma that are involved in immune suppression will lead to new therapeutic opportunities. One stromal protein, βig-h3 (also known as TGFβi), has been shown to be associated with poor prognosis in pancreatic ductal adenocarcinoma (PDAC) and other cancers (e.g., lung, head and neck, colorectal, bladder, and melanoma) when overexpressed in the stroma.

[0003] Exploring the potential role of the βig-h3 stromal protein in PDAC models (both in mice and humans) has revealed that this protein reduces the cytotoxic activity of T lymphocytes and helps stiffen the microenvironment, making the tumor less accessible to the immune system.

[0004] In mice and humans, βig-h3 protein is not expressed in the pancreatic exocrine compartment of healthy individuals but appears very early in the tumor stroma.

[0005] It has been proposed that targeting some of the key components (such as βig-h3) with specific drugs could alter the tumor stroma and aid in the therapeutic treatment of solid tumors. It has been proposed that specific depletion of this protein could restore CD8+ T cell activity and reduce stromal stiffness, thus restoring access to the tumor.

[0006] One standard approach to depleting a protein is to generate monoclonal antibodies (mAbs) that are specifically directed against key epitopes and thereby block the functional activity of that protein, and the protein / antibody complex is then eliminated from the body.

[0007] Antibodies against the βig-h3 protein have been shown to play a role in directly altering the antitumor immune response by blocking the inhibition of CD8+ T cell activation (WO 2017 / 158043). A mouse monoclonal antibody directed against the human βig-h3 protein (called 18B3) was described in WO 2020 / 079164. Summary of the Invention

[0008] There is a continuing need for new and preferably improved means of cancer treatment. Thus, one object of the present invention is to provide improved means for treating cancer. In particular, it is envisaged that these improved means will specifically deplete βig-h3 protein, restore CD8+ T cell activity, and reduce stromal stiffness, thereby restoring or facilitating immune system access to tumors, ultimately leading to significant tumor shrinkage and survival rates. It is also envisaged to facilitate drug access to tumors. The present approach to deplete βig-h3 protein is to generate humanized monoclonal antibodies (mAbs) that are specifically directed against key epitopes to block the functional activity of the protein. The protein / antibody complex is then cleared from the body.

[0009] The present invention therefore relates to humanized (Hz) antibodies with specificity against the βig-h3 protein and their uses. These Hz antibodies are βig-h3 antagonists. In particular, the invention is defined by the claims. These antibodies are humanized versions of the 18B3 antibody. These humanized antibodies have particularly attractive and unexpected properties (e.g. affinity, dissociation rate, thermostability, productivity in cell culture) that make them promising antibodies for therapeutic use and in particular for restoring the activity of CD8+ T cells and reducing stromal stiffness, thus restoring or facilitating the access of the immune system and / or drugs to the tumor. These humanized mAbs have therefore proven successful in in vitro and in vivo functional bioassays.

[0010] The mAb targets a region of the βig-h3 protein known to be involved in binding to integrins (involved in T cell activation pathways) and collagens (involved in tumor microenvironment or stromal changes) on the surface. This region is expressed by avb3 ( αVβ3 ) integrin interaction motif, present in the fragment corresponding to amino acids (AA) 548-614. Epitope mapping studies showed that the antibody targets the fourth domain of FAS1 (linear epitope ALPPRERSRL, SEQ ID NO: 16, which can even be shortened to the central 8 amino acids, SEQ ID NO: 30) (AA residues 549-558) of the βig-h3 protein. Several affinity and functional bioassays reported herein can confirm the specific binding of the humanized (Hz) monoclonal antibody disclosed herein.

[0011] In one aspect, the present invention provides a method for the treatment of a βig-h3 protein epitope, the epitope being set forth in SEQ ID NO: 16 or SEQ ID NO: 30, with a high affinity K of 1 nM or less, preferably 0.7 nM or less, more preferably 0.6 or 0.58 nM or less, as measured by surface plasmon resonance (SPR). D and 4 and 10E-04 seconds-1 Between 5 and 8E-04 seconds, preferably -1 Between 5.5 and 7.5E-04 seconds, more preferably -1 The slow dissociation rate K d The present invention relates to a humanized anti-βig-h3 monoclonal antibody or an antigen-binding fragment thereof that specifically binds to SPR. SPR can be measured using a biosensor system (such as a Biacore® system).

[0012] The affinities and dissociation rates disclosed herein were measured as described in the Methods section.

[0013] In one embodiment, the present invention relates to a humanized anti-βig-h3 monoclonal antibody or antigen-binding fragment thereof, comprising a variable domain VH and a variable domain VL, specifically binding to an epitope of the βig-h3 protein. Said epitope is shown as sequence SEQ ID NO: 16 or 30. The VH domain has a sequence shown as sequence SEQ ID NO: 4 or 28. SEQ ID NO: 28 is a mutated version of SEQ ID NO: 4, i.e., cysteine ​​102 in H-CDR3 is replaced by serine. The VL is a humanized variant of the mouse 18B3 VL domain, and has a sequence shown as SEQ ID NO: 18. This combination of VH and VL provides the humanized anti-βig-h3 monoclonal antibody or antigen-binding fragment thereof with a large and unexpected thermostability, i.e. a DSC of above 80° C., in particular comprised between 80 and 83, 83.2, 83.5, or 84° C., preferably between 81 and about 83 or 83.2° C. The DSC is measured using the method described in the Methods of Measurement section. In one aspect, the humanized anti-βig-h3 monoclonal antibody or antigen-binding fragment thereof further comprises a high affinity K for the epitope of 1 nM or less, preferably 0.7 nM or less, more preferably 0.6 or 0.58 nM or less, as measured using surface plasmon resonance (SPR). D , and / or 4 and 10E-04 seconds -1 Between 5 and 8E-04 seconds, preferably -1 Between 5.5 and 7.5E-04 seconds, more preferably -1 The slow dissociation rate Kd SPR can be measured using a biosensor system (such as a Biacore® system).

[0014] The humanized antibodies of the present invention are capable of depleting βig-h3 protein.

[0015] The humanized antibodies of the present invention can restore the activity of CD8+ T cells and / or reduce stromal stiffness, thereby restoring access to the tumor, and therefore these antibodies can be used in combination with other anti-tumor agents that can more easily access the tumor thanks to the effect of anti-βig-h3 antibodies on the stroma.

[0016] The present invention also relates to a pharmaceutical composition comprising at least one humanized monoclonal antibody or antigen-binding fragment thereof and a pharma- ceutically acceptable vehicle.

[0017] The present invention also relates to a pharmaceutical composition, pharmaceutical combination, or kit of parts comprising at least one humanized monoclonal antibody or antigen-binding fragment thereof and another anti-tumor agent (eg, an antibody, in particular a monoclonal antibody or fragment thereof).

[0018] The invention also relates to such antibodies, pharmaceutical compositions, pharmaceutical combinations, or kits of parts for use in preventing or treating cancer, depleting βig-h3 protein, restoring or activating CD8+ T cell activity, and / or reducing stromal stiffness to facilitate access of other anti-tumor agents (antibodies, monoclonal antibodies, etc.) to the tumor.

[0019] The invention also relates to a method of preventing or treating cancer, comprising administering to a patient in need thereof an effective amount of such an antibody, pharmaceutical composition, pharmaceutical combination, or kit of parts. The invention also relates to depleting βig-h3 protein, restoring or activating CD8+ T cell activity, and / or reducing stromal stiffness to facilitate access of other anti-tumor agents (antibodies, monoclonal antibodies, etc.) to the tumor. [Brief description of the drawings]

[0020] [Figure 1] Figure 1 is a schematic diagram of the structure of human βig-h3. 18B3 mAb recognizes the 549-558 epitope, which contains the YH18 domain that is important for the binding of αvβ3 to collagen. [Diagram 2] Figure 2 is a graph showing the results of the ELISA for chimeric 18B3 (considered as the reference mAb) and the four humanized variants. Mean (+ standard deviation) of six experiments. [Diagram 3] Figure 3: Graph showing cytotoxic CD8+ T cell activation and proliferation for chimeric 18B3 (considered as reference mAb) and the four humanized variants. Mean (+ standard deviation) of three experiments. Statistical significance of parameters is assessed through Student's t-test and one-way analysis of variance performed using GraphPad Prism software. [Figure 4]Figure 4 is a graph showing the tumor weight of subcutaneously implanted tumor pancreatic cancer tumor cells in the presence of ctrl IgG1 Ab, chimeric 18B3 (considered as reference mAb), as well as the four humanized variants. Tumor cells are embedded as clusters in a Matrigel 1:1 mixture (Corning) and injected subcutaneously into the flank of normal C57BL6 mice with 6 mg of humanized version mAb per mouse. The control is an irrelevant isotype control IgG1 mAb. The same mouse population (n=5) is used for each dose evaluated. The tumor grafts are isolated and the amount of tumor cells within the grafts is then evaluated at 4°C by FACS staining and analyzed with FlowJo software. The statistical significance of the parameters is evaluated through Student's t-test and one-way analysis of variance performed with GraphPad Prism software. [Diagram 5] Figure 5 is a graph showing the number of tumor cells in subcutaneous grafts in the presence of ctrl IgG1 Ab, chimeric 18B3 (considered as reference mAb), as well as the four humanized variants. Tumor cells are embedded as clusters in a Matrigel 1:1 mixture (Corning) and injected subcutaneously into the flank of normal C57BL6 mice with 6 mg of humanized version mAb per mouse. The control is an irrelevant isotype control IgG1 mAb. The same mouse population (n=5) is used for each dose evaluated. The tumor grafts are isolated and the amount of tumor cells in the grafts is then evaluated at 4°C by FACS staining and analyzed with FlowJo software. The statistical significance of the parameters is evaluated through Student's t-test and one-way analysis of variance performed with GraphPad Prism software. [Figure 6]Figure 6 is a graph showing the number of non-activated CD8 T cells in subcutaneous grafts in the presence of ctrl IgG1 Ab, chimeric 18B3 (considered as reference mAb), as well as the four humanized variants. Tumor cells are embedded as clusters in a Matrigel 1:1 mixture (Corning) and injected subcutaneously into the flank of normal C57BL6 mice with 6 mg of humanized version mAb per mouse. The control is an irrelevant isotype control IgG1 mAb. The same mouse population (n=5) is used for each dose evaluated. The tumor grafts are isolated and the amount of tumor cells in the grafts is then evaluated at 4°C by FACS staining and analyzed with FlowJo software. The statistical significance of the parameters is evaluated through Student's t-test and one-way analysis of variance performed with GraphPad Prism software. [Figure 7] Figure 7 is a graph showing the number of tumor cells in subcutaneous grafts in the presence of ctrl IgG1 Ab as well as the original version (V1) and the mutated version for C102 (V1.2) of the two humanized variants (H330 / L41 and H330 / L228). A mutation at position 102 of the heavy chain 330 (substitution of a cysteine ​​residue by a serine) is realized for both mAbs to minimize the risk of post-translational modifications (PTM). The tumor cells are embedded as clusters in a Matrigel 1:1 mixture (Corning) and injected subcutaneously into the flank of normal C57BL6 mice together with 6 mg of the humanized version mAb per mouse. The control is an irrelevant isotype control IgG1 mAb. The same mouse population (n=5) is used for each dose evaluated. The tumor grafts are dissociated and the amount of tumor cells in the grafts is then evaluated at 4°C by FACS staining and analyzed with FlowJo software. The statistical significance of parameters is assessed through Student's t-test and one-way analysis of variance performed using GraphPad Prism software. [Figure 8]Figure 8 is a graph showing the number of activated CD8 T cells in subcutaneous grafts in the presence of ctrl IgG1 Ab as well as the original version (V1) of the two humanized variants (H330 / L41 and H330 / L228) and the mutated version for C102 (V1.2). Tumor cells are embedded as clusters in a Matrigel 1:1 mixture (Corning) and injected subcutaneously into the flank of normal C57BL6 mice with 6 mg of humanized version mAb per mouse. The control is an irrelevant isotype control IgG1 mAb. The same mouse population (n=5) is used for each dose evaluated. The tumor grafts are isolated and the amount of tumor cells in the grafts is then evaluated at 4°C by FACS staining and analyzed with FlowJo software. The statistical significance of the parameters is evaluated through Student's t-test and one-way analysis of variance performed with GraphPad Prism software. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] Humanized antibodies

[0022] In one aspect, the present invention relates to a humanized anti-βig-h3 monoclonal antibody or antigen-binding fragment thereof, comprising a variable domain VH and a variable domain VL, which specifically binds to an epitope of the βig-h3 protein. The epitope is preferably as shown in sequence SEQ ID NO: 16 or 30. Of course, it cannot be excluded that an antibody or a fragment thereof of the present invention is capable of binding to a βig-h3 protein fragment larger than SEQ ID NO: 16 or 30 and to a βig-h3 protein fragment comprising this sequence. The binding of said antibody or a fragment thereof occurs with a surprisingly high level of affinity, in particular a high affinity K of ≦1 nM, preferably ≦0.7 nM, more preferably ≦0.6 or 0.58 nM, as measured using surface plasmon resonance (SPR). D Remarkably and unexpectedly, the Hz antibody or fragment thereof exhibits a slow dissociation rate, K, after binding. dThe slow dissociation rates are between 4 and 10E-04 seconds when measured using SPR. -1 Between 5 and 8E-04 seconds, preferably -1 Between 5.5 and 7.5E-04 seconds, more preferably -1 SPR can be measured using a biosensor system (such as a Biacore® system).

[0023] The sequences of interest in this application are shown in Table 1 below:

[0024] [Table 1-1] [Table 1-2] [Table 1-3] Legend: In VL and VH, CDRs according to Kabat are in bold, other amino acids are in FR, and special amino acids that differ from mouse are underlined.

[0025] Two humanized VH domains (H-330 and H-311) and two humanized VL domains (L-41 and L-228) were generated, each of which is an object of the present invention, as are their VH / VL combinations in monoclonal antibodies or fragments thereof. The humanized VH domain H-169 and the humanized VL domain L-315 are also objects of the present invention, as are their VL or VH domains and their combinations in monoclonal antibodies or fragments thereof.

[0026] A mutated version of H-330 was also generated with a mutation of cysteine ​​102 to serine, designated H330V1.2 (versus V1 for the original H330) or H330C102S, and has the sequence shown in SEQ ID NO: 28. This mutation occurs in H-CDR-3, and the mutated H-CDR3 has the sequence shown in SEQ ID NO: 27.

[0027] These VH domains, including those that are mutated, can be combined to design the antibody binding domains H-330 / L-41, H-330 / L-228, H-330V1.2 / L-41, H-330V1.2 / L-228, H-311 / L-41, and H-311 / L-228, each of which is an object of the present invention. The humanized VH domains H-330, H-330V1.2, and H-311, particularly the domains H-330 and H-330V1.2, can also be combined with any humanized VL domain derived from the m18B3 monoclonal antibody. In particular, the humanized VL domain comprises L-CDR1, L-CDR2 and L-CDR3 of the sequences shown in SEQ ID NO: 7, 8 and 9, respectively; or L-CDR1, L-CDR2 and L-CDR3 of the sequences shown in SEQ ID NO: 11, 12 and 9, respectively. These combinations are believed to specifically bind to the βig-h3 protein, more particularly to the fourth domain of FAS1 of the βig-h3 protein (epitopes shown in SEQ ID NO: 16 or 30) (AA residues 549-558) of the βig-h3 protein. The methods described herein, in particular SPR methods, such as biosensor systems (such as the Biacore® system), can be used to confirm the binding with good or very high affinity as described above, and to determine the suitability of candidates comprising domains H-330, H-330V1.2 or H-311 and humanized domains derived from m18B3. The dissociation rates can be determined in the same manner to determine the suitability of these candidates.

[0028] The humanized variants have no statistically significant difference compared to chimeric 18B3 in terms of affinity measured by ELISA, indicating that the humanization process did not change the affinity measured by ELISA. Using the Biacore® system, all these humanized mAbs have affinities (KD) in the sub-nanomolar range and surprisingly have slower off-rates than mouse 18B3 and chimeric 18B3. The humanized variant H-330 / L-228 shows the highest affinity of the humanized mAbs.

[0029] Thus, in some aspects, the invention relates to humanized anti-βig-h3 monoclonal antibodies or antigen-binding fragments thereof, including any of H-330 / L-41, H-330 / L-228, H-330V1.2 / L-41, H-330V1.2 / L-228, H-311 / L-41, or H-311 / L-228. These antibodies or antigen-binding fragments thereof specifically bind to an epitope of the βig-h3 protein, said epitope being set forth as sequence SEQ ID NO: 16 or 30 (or a longer sequence as described above). This binding is particularly with a high affinity K of 1 nM or less, preferably 0.7 nM or less, more preferably 0.6 or 0.58 nM or less, as measured using SPR. D This binding occurs at 4 and 10E-04 seconds, as measured using SPR. -1 Between 5 and 8E-04 seconds, preferably -1 Between 5.5 and 7.5E-04 seconds, more preferably -1 The slow dissociation rate K d Conveniently, SPR occurs at 100° C. SPR can be measured using a biosensor system (such as a Biacore® system).

[0030] H-330 and H-330V1.2, as exemplified by combinations with all L-variants examined (either L-41 or L-228), provide the monoclonal antibody with an unexpectedly high thermal stability according to the DSC, which is above 80°C, specifically between 81 and 83, 83.2, or 83.5°C. Additional data shown in the Examples with other combinations of VH and VL domains indicate that H-330 is important for this improved thermal stability, whatever the complementary VH domain. The thermal stability of H-330V1.2 with the mutation C102S remains largely above 80°C. H-330 and H-330V1.2, as exemplified by combinations with all L-variants examined (either L-41 or L-228), also provide the monoclonal antibody with an unexpectedly high productivity in transient expression in CHO cells. Its productivity is above 200 μg / ml, specifically comprised between 230 and 300 μg / ml. This is accompanied by a very good affinity by ELISA and Biacore®, with the highest Biacore® affinity for the humanized variant H-330 / L-228. These special properties, linked to the presence of H-330 or its mutated version C102S in the monoclonal antibody, are surprising when compared to the results obtained with H-311 used in combination with the same VL variant, with only six amino acid differences between H-330 and H-311.

[0031] The heavy chain H-330, in its V1 version (SEQ ID NO: 4) or in its C102S V1.2 mutated version (SEQ ID NO: 28), when associated with different light chains (L-41, L-228, L-315), shows great and unexpected thermostability among the monoclonal antibodies disclosed herein that bind to the epitope of the βig-h3 protein (epitope of sequence SEQ ID NO: 16 or 30). The variant with the cysteine ​​to serine mutation at position 102 of the heavy chain showed similar conserved reactivity and stability properties as the non-mutated mAb version (H-330V1), and is therefore a valuable candidate for druggability purposes.

[0032] The DSCs disclosed herein and transient expression in CHO were assayed as described in the Methods of Assay.

[0033] In a particular aspect, the present invention provides a method for the treatment of βig-h3 protein, preferably the epitope shown in sequence SEQ ID NO: 16 or 30, with a high affinity K of 1 nM or less, preferably 0.7 nM or less, more preferably 0.6 or 0.58 nM or less, as measured by SPR, in particular with the above-mentioned binding affinity and / or dissociation rate. D ;When measured using SPR, especially 4 and 10E-04 seconds -1 Between 5 and 8E-04 seconds, preferably -1 Between 5.5 and 7.5E-04 seconds, more preferably -1 The slow dissociation rate K d ) and specifically binds to (a) - H-CDR1 having the sequence shown as SEQ ID NO:1; - H-CDR2 having the sequence shown as SEQ ID NO:2; - a variable domain VH comprising a H-CDR3 (comprising the CDRs of H-330 variant or H-330V1.2) with the sequence as shown in SEQ ID NO: 3 or 27; (b) A humanized anti-βig-h3 monoclonal antibody or an antigen-binding fragment thereof, which is a humanized variant of the 18B3 monoclonal antibody, i.e., comprises a variable domain VL which is a humanized variant of the 18B3 VL domain having the sequence shown as SEQ ID NO: 18.

[0034] In one embodiment, the antibody comprises a VH domain having the sequence shown as SEQ ID NO:4.

[0035] The monoclonal antibody or antigen-binding fragment thereof specifically binds to βig-h3 protein, and more particularly to the fourth domain of FAS1 of βig-h3 protein (the epitope shown as SEQ ID NO: 16 or 30), with a high affinity K of ≦1 nM, preferably ≦0.7 nM, and more preferably ≦0.6 or 0.58 nM, as measured using SPR. D and 4 and 10E-04 seconds -1 Between 5 and 8E-04 seconds, preferably -1 Between 5.5 and 7.5E-04 seconds, more preferably -1 The slow dissociation rate K d SPR can be measured using a biosensor system (such as a Biacore® system).

[0036] In another particular aspect, the present invention provides an antibody that specifically binds to βig-h3 protein and (a) - H-CDR1 having the sequence shown as SEQ ID NO:1; - H-CDR2 having the sequence shown as SEQ ID NO:2; - a variable domain VH comprising a H-CDR3 (comprising the CDRs of H-330 variant or H-330V1.2) with the sequence as shown in SEQ ID NO: 3 or 27; (b)-L-CDR1 having the sequence shown as SEQ ID NO:7; - L-CDR2 having the sequence shown as SEQ ID NO: 8; - L-CDR3 having the sequence shown as SEQ ID NO: 9 The present invention relates to a humanized anti-βig-h3 monoclonal antibody or an antigen-binding fragment thereof comprising a variable domain VL (including the CDRs of the L-41 variant).

[0037] In one embodiment, the antibody comprises a VH domain having the sequence shown as SEQ ID NO: 4 or 28, and / or a VL domain having the sequence shown as SEQ ID NO: 10.

[0038] The monoclonal antibody or antigen-binding fragment thereof is - K of about 5.8E-10M or less, in particular between about 5E-10 and about 5.8E-10M or less, in particular about 5.35E-10M D and / or - Approx. 6E-04 seconds -1 Above, especially about 6.2E-04 and about 7E-04 seconds -1 Especially at about 6.59E-04 seconds -1 K d and / or - have a stability of about 79°C or higher, in particular between about 79 and about 83, 83.2, or 83.5°C, typically about 81.3°C, as measured by DSC (Tm Fab); and / or - Excellent productivity measured at approximately 275 μg / ml in transient expression in CHO cells.

[0039] In one embodiment, the humanized anti-βig-h3 monoclonal antibody (H-330 / L-41 or H-330V1.2 / L-41) or antigen-binding fragment thereof is - a VH domain having the sequence as set forth in SEQ ID NO: 4 or 28; - comprises a VL domain having the sequence shown as SEQ ID NO: 10.

[0040] In one embodiment, the humanized anti-βig-h3 antibody is - a heavy chain comprising said variable domain and a constant domain CH (such as the CH having the sequence shown as SEQ ID NO: 14); - a light chain comprising said variable domain and a constant domain CL (such as CL having the sequence given as SEQ ID NO: 15).

[0041] In another particular aspect, the present invention provides an antibody that specifically binds to βig-h3 protein and (a) - H-CDR1 having the sequence shown as SEQ ID NO:1; - H-CDR2 having the sequence shown as SEQ ID NO:2; - a variable domain VH comprising a H-CDR3 (comprising the CDRs of H-330 variant or H-330V1.2) with the sequence as shown in SEQ ID NO: 3 or 27; (b)-L-CDR1 having the sequence shown as SEQ ID NO:11; - L-CDR2 having the sequence shown as SEQ ID NO: 12; - L-CDR3 having the sequence shown as SEQ ID NO: 9 The present invention relates to a humanized anti-βig-h3 monoclonal antibody or an antigen-binding fragment thereof comprising a variable domain VL (including the CDRs of the L-228 variant).

[0042] In one embodiment, the antibody comprises a VH domain having the sequence shown as SEQ ID NO: 4 or 28, and / or a VL domain having the sequence shown as SEQ ID NO: 13.

[0043] The monoclonal antibody or antigen-binding fragment thereof is - K of about 5E-10M or less, in particular between about 4.5E-10M and about 5E-10M, typically about 4.76E-10M D and / or - About 5E-04 seconds -1 Above, especially about 5.5E-04 and about 6E-04 seconds -1 Especially at about 5.83E-04 seconds -1 K d and / or - have a stability of about 78°C or higher, particularly about 78-82°C, typically about 80.2°C, as measured by DSC (Tm Fab); and / or - Excellent productivity measured at approximately 249 μg / ml in transient expression in CHO cells.

[0044] In one embodiment, the humanized anti-βig-h3 monoclonal antibody (H-330 / L-228 or H-330V1.2 / L-228) or antigen-binding fragment thereof is - a VH domain having the sequence as set forth in SEQ ID NO: 4 or 28; - comprises a VL domain having the sequence shown as SEQ ID NO: 13.

[0045] In one embodiment, the humanized anti-βig-h3 monoclonal antibody is - a heavy chain comprising said variable domain and a constant domain CH (such as the CH having the sequence shown as SEQ ID NO: 14); - a light chain comprising said variable domain and a constant domain CL (such as CL having the sequence given as SEQ ID NO: 15).

[0046] In another particular aspect, the present invention provides an antibody that specifically binds to βig-h3 protein and (a) - H-CDR1 having the sequence shown as SEQ ID NO:1; - H-CDR2 having the sequence shown as SEQ ID NO:5; - a variable domain VH comprising H-CDR3 (comprising the CDRs of the H-311 variant) with the sequence shown as SEQ ID NO: 3; (b)-L-CDR1 having the sequence shown as SEQ ID NO:7; - L-CDR2 having the sequence shown as SEQ ID NO: 8; - L-CDR3 having the sequence shown as SEQ ID NO: 9 The present invention relates to a humanized anti-βig-h3 monoclonal antibody or an antigen-binding fragment thereof comprising a variable domain VL (including the CDRs of the L-41 variant).

[0047] In one embodiment, the antibody comprises a VH domain having the sequence shown as SEQ ID NO:6 and / or a VL domain having the sequence shown as SEQ ID NO:10.

[0048] The monoclonal antibody or antigen-binding fragment thereof is - K less than or equal to about 5E-10M, in particular between about 4.5 and about 5E-10M, in particular about 4.82E-10M D and / or - About 6.8E-04 seconds -1Above, especially about 7E-04 and about 7.5E-04 seconds -1 Especially at about 7.26E-04 seconds -1 K d and / or - have a stability such that the DSC (Tm Fab) is greater than about 75°C, in particular between about 75 and about 79°C, typically about 77°C.

[0049] In one embodiment, the humanized anti-βig-h3 monoclonal antibody (H-311 / L-41) or antigen-binding fragment thereof is - a VH domain having the sequence shown as SEQ ID NO:6; - comprises a VL domain having the sequence shown as SEQ ID NO: 10.

[0050] In one embodiment, the humanized anti-βig-h3 antibody is - a heavy chain comprising said variable domain and a constant domain CH (such as the CH having the sequence shown as SEQ ID NO: 14); - a light chain comprising said variable domain and a constant domain CL (such as CL having the sequence given as SEQ ID NO: 15).

[0051] In another particular aspect, the present invention provides an antibody that specifically binds to βig-h3 protein and (a) - H-CDR1 having the sequence shown as SEQ ID NO:1; - H-CDR2 having the sequence shown as SEQ ID NO:5; - a variable domain VH comprising H-CDR3 (comprising the CDRs of the H-311 variant) with the sequence shown as SEQ ID NO: 3; (b)-L-CDR1 having the sequence shown as SEQ ID NO:11; - L-CDR2 having the sequence shown as SEQ ID NO: 12; - L-CDR3 having the sequence shown as SEQ ID NO: 9 The present invention relates to a humanized anti-βig-h3 monoclonal antibody or an antigen-binding fragment thereof comprising a variable domain VL (including the CDRs of the L-228 variant).

[0052] In one embodiment, the antibody comprises a VH domain having the sequence shown as SEQ ID NO:6 and / or a VL domain having the sequence shown as SEQ ID NO:13.

[0053] The monoclonal antibody, or antigen-binding fragment thereof, - K of about 5E-10M or less, in particular between about 4.5 and about 5E-10M, in particular about 4.9E-10M D and / or - About 6.5E-04 seconds -1 Above, especially 6.8E-04 and 7.3E-04 seconds -1 Especially at about 7.07E-04 seconds -1 K d and / or - have a stability of DSC (Tm Fab) of about 73.5°C or higher, in particular of about 73.5 and about 77.5°C, typically about 75.5°C.

[0054] In one embodiment, the humanized anti-βig-h3 monoclonal antibody (H-311 / L-228) or antigen-binding fragment thereof is - a VH domain having the sequence shown as SEQ ID NO:6; - comprises a VL domain having the sequence shown as SEQ ID NO: 13.

[0055] In one embodiment, the humanized anti-βig-h3 monoclonal antibody is - a heavy chain comprising said variable domain and a constant domain CH (such as the CH having the sequence shown as SEQ ID NO: 14); - a light chain comprising said variable domain and a constant domain CL (such as CL having the sequence given as SEQ ID NO: 15).

[0056] In one embodiment the humanized antibody disclosed herein comprises a human IgG1 constant domain, preferably the Constant domain human for Heavy chain Heavy Human IgG1 m1,17 of SEQ ID NO:14, and / or a constant domain of the light chain, in particular kappa, preferably the Constant domain human for light chain (kappa) - light chain human Km3 of SEQ ID NO:15.

[0057] Definition and characteristics

[0058] Residues in antibody variable domains are usually numbered according to a system devised by Kabat et al., which is set forth in Kabat et al., 1987, Sequences of Proteins of Immunological Interest, United States Department of Health and Human Services, NIH, United States (hereafter "Kabat et al."). This numbering system is used herein. The Kabat residue designations do not necessarily correspond to the linear numbering of the amino acid residues in the sequence of SEQ ID NO:. The actual linear amino acid sequence may contain fewer or additional amino acids than in the strict Kabat numbering, which corresponds to truncation of, or insertion into, structural elements of the basic variable domain structure, whether framework or complementarity determining regions (CDRs). The correct Kabat numbering of residues can be determined for a given antibody by alignment of homologous residues in the sequence of the antibody with the "standard" Kabat numbered sequence. The CDRs of the heavy chain variable domain are located at residues 31-35B (H-CDR1), residues 50-65 (H-CDR2), and residues 95-102 (H-CDR3) according to the Kabat numbering system. The CDRs of the light chain variable domain are located at residues 24-34 (L-CDR1), residues 50-56 (L-CDR2), and residues 89-97 (L-CDR3) according to the Kabat numbering system (http: / / www.bioinf.org.uk / abs / #cdrdef).

[0059] The term "antigen-binding fragment" of an antibody herein refers to one or more fragments of an intact antibody that retain the ability to specifically bind to the βig-h3 antigen. The antigen-binding function of an antibody can be performed by a fragment of an intact antibody. Examples of binding fragments that fall within the scope of the term antigen-binding fragment of an antibody include Fab fragments (monovalent fragments consisting of the VL, VH, CL, and CH1 domains); Fab' fragments (monovalent fragments consisting of the VL, VH, CL, CH1 domains, and hinge region); F(ab')2 fragments (bivalent fragments containing two Fab' fragments linked by a disulfide bridge at the hinge region); Fd fragments consisting of the VH domain of a single arm of an antibody; single domain antibody (sdAb) fragments (Ward et al., 1989 Nature 341:544-546) (which consist of the VH or VL domain); and isolated complementarity determining regions (CDRs). Furthermore, although the two domains (VL and VH) of the Fv fragment are encoded by separate genes, they can be joined by an artificial peptide linker using recombinant techniques, which allows the pairing of the VL and VH regions into a single protein chain forming a monovalent molecule (known as single-chain Fv (ScFv); see, for example, Bird et al., 1989 Science 242:423-426 and Huston et al., 1988 proc. Natl. Acad. Sci. 85:5879-5883). A "dsFv" is a VH::VL heterodimer stabilized by a disulfide bond. Bivalent and multivalent antibody fragments can be formed spontaneously by association of monovalent scFvs or can be generated by coupling monovalent scFvs with a peptide linker (such as a bivalent sc(Fv)2). Such single-chain antibodies contain one or more antigen-binding portions or fragments of an antibody. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as intact antibodies. Unibodies are another type of antibody fragment that lacks the hinge region of IgG4 antibodies.The deletion of the hinge region results in a molecule that is essentially half the size of a traditional IgG4 antibody and has a monovalent binding region rather than the bivalent binding region of IgG4 antibodies. Antigen-binding fragments can be assembled into single domain antibodies, SMIPs, maxibodies, minibodies, intrabodies, diabodies, triabodies, and tetrabodies (see, for example, Hollinger and Hudson, 2005, Nature Biotechnology, 23, 9, 1126-1136). The terms "diabody", "triabody" or "tetrabody" refer to small antibody fragments with multiple antigen-binding sites (2, 3, or 4), which comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same peptide chain (VH-VL). By using a linker that is too short to allow pairing between these two domains on the same chain, these domains are forced to pair with complementary domains on another chain, creating two antigen-binding sites. The antigen-binding fragment can be assembled into a single chain molecule comprising a pair of tandem Fv segments (VH-CH1-VH-CH1), which associates with complementary light chain polypeptides to form a pair of antigen-binding regions (Zapata et al., 1995 Protein Eng. 8(10); 1057-1062 and U.S. Pat. No. 5,641,870).

[0060] In one embodiment, the antibody fragment of the invention is an antigen-binding fragment selected from the group consisting of Fab, F(ab)'2, single domain antibody, ScFv, Sc(Fv)2, diabody, triabody, tetrabody, unibody, minibody, maxibody, small modular immunopharmaceutical (SMIP), minimal recognition unit consisting of amino acid residues mimicking a hypervariable region of an antibody as an isolated complementarity determining region (CDR), and fragments comprising or consisting of a VL domain or a VH domain as disclosed herein.

[0061] The Fab of the present invention can be obtained by treating an antibody that specifically reacts with βig-h3 with the protease papain. The Fab can also be produced by inserting DNA encoding the Fab of the antibody into a vector for a prokaryotic or eukaryotic expression system and introducing the vector into a prokaryote or eukaryote (as needed) to express the Fab.

[0062] The F(ab')2 of the present invention can be obtained by treating an antibody that specifically reacts with βig-h3 with the protease pepsin. F(ab')2 can be produced by linking the following Fab' via a thioether bond or disulfide bond.

[0063] The Fab' of the present invention can be obtained by treating F(ab')2 that specifically reacts with βig-h3 with a reducing agent, dithiothreitol. Alternatively, the Fab' can be produced by inserting DNA encoding the Fab' fragment of an antibody into an expression vector for prokaryotes or eukaryotes, and introducing the vector into a prokaryote or eukaryote (as required) for expression.

[0064] The scFv of the present invention can be produced by obtaining cDNAs encoding the VH and VL domains as previously described, constructing DNA encoding the scFv, introducing the DNA into an expression vector for prokaryotes or an expression vector for eukaryotes, and then introducing the expression vector into a prokaryote or eukaryote (as appropriate) to express the scFv. A well-known technique called CDR grafting can be used to produce humanized scFv fragments. This technique involves selecting complementarity determining regions (CDRs) from a donor scFv fragment and grafting them onto a human scFv fragment framework of known three-dimensional structure (see, for example, WO 98 / 45322; WO 87 / 02671; US ​​Patent No. 5,859,205; US Patent No. 5,585,089; US Patent No. 4,816,567; EP Patent No. 0173494).

[0065] The humanized monoclonal antibody of the present invention can be produced by obtaining nucleic acid sequences encoding the CDR domains as previously described, inserting them into an expression vector for animal cells having genes encoding (i) the same heavy chain constant region as a human antibody and (ii) the same light chain constant region as a human antibody to construct a humanized antibody expression vector, and expressing the genes by introducing the expression vector into the animal cell. The humanized antibody expression vector can be either a type in which the gene encoding the antibody heavy chain and the gene encoding the antibody light chain are present on separate vectors, or a type in which both genes are present on the same vector (tandem type). In terms of the ease of constructing the humanized antibody expression vector, the ease of introducing it into animal cells, and the balance between the expression levels of the antibody H chain and L chain in the animal cell, the tandem type humanized antibody expression vector is preferred. Examples of tandem type humanized antibody expression vectors include pKANTEX93 (WO 97 / 10354), pEE18, etc. Methods for producing humanized antibodies based on conventional recombinant DNA and gene transfection techniques are well known in the art (see, for example, Riechmann L. et al. 1988; Neuberger MS. et al. 1985). Antibodies can be humanized using a variety of techniques known in the art, including, for example, CDR-grafting (European Patent No. 239,400; PCT Publication No. WO 91 / 09967; U.S. Patent Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (European Patent No. 592,106; European Patent No. 519,596; Padlan EA (1991); Studnicka GM et al. (1994); Roguska MA. et al. (1994)), and chain shuffling (U.S. Patent No. 5,565,332). General recombinant DNA techniques for preparing such antibodies are also known (see European Patent Application EP 125023 and International Patent Application WO 96 / 02576).

[0066] In this specification, the symbol K Dis assumed to mean the dissociation constant, which is obtained from the ratio of Kd to Ka (i.e., Kd / Ka) and is expressed as a molar concentration (M). D The value can be determined using methods well established in the art. D One way to determine is by using SPR, in particular a biosensor system (such as a Biacore® system) under the conditions described in the method of measurement section.

[0067] The symbol "k d ”(seconds -1 ) refers herein to the dissociation rate constant of a particular Ab-antigen interaction ([Ab]][antigen] / [Ab-antigen complex]). off Also called value.

[0068] The symbol "k a " " -1 × seconds -1 ) refers herein to the association rate constant of a particular Ab-antigen interaction, and k d is the reciprocal of.

[0069] The symbol "K D " (M)" refers herein to the dissociation equilibrium constant of a particular Ab-antigen interaction, k d k a It is obtained by dividing by.

[0070] The symbol "K A " " -1 ) is used herein to refer to the association equilibrium constant of a particular Ab-antigen interaction, and k a k d It is obtained by dividing by.

[0071] Herein, thermal stability is assessed by differential scanning calorimetry (DSC), which is measured as described in the Methods of Measurement section.

[0072] By "humanized antibody" or "chimeric antibody" is meant an antibody derived from a parent murine antibody by methods available to one of skill in the art, for example as disclosed herein. Preferably, the "humanized antibody" or "chimeric antibody", or an antigen-binding fragment thereof, comprises a set of six CDRs of the murine antibody m18B3, possibly with mutations within the CDRs.

[0073] Humanized antibodies (such as chimeric antibodies) and antigen-binding fragments retain or substantially retain the antigen-binding properties of the parent murine antibody m18B3, and as disclosed herein, humanization can impart interesting and unexpected functionalities to murine and / or chimeric 18B3 monoclonal antibodies.

[0074] The CDRs, or some of them, may differ from the mouse CDRs after the SDR approach (hypergrafting) or other useful methods. The humanization described herein makes it possible to provide monoclonal antibodies and antigen-binding fragments thereof with interesting and unexpected functions (particularly affinity, dissociation rate, thermal stability (DSC)) combined with therapeutic functional properties, as disclosed herein. H-330 and H-311 proved to be very attractive, as did L-41 and L-218. The skilled person will be able to introduce amino acid changes (e.g. up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acids) into these VH and VL regions without substantially altering some of their functionality and functional properties. Such altered VH and / or VL domains are also considered to be encompassed in the definition of these VH and VL domains.

[0075] The various antibody molecules and fragments can be derived from any of the commonly known immunoglobulin classes, non-limiting examples of which include IgA, secretory IgA, IgE, IgG, and IgM. IgG subclasses are also well known in the art, non-limiting examples of which include human IgG1, IgG2, IgG3, and IgG4. It is preferred to use IgG1.

[0076] "Treatment" or "therapy" refers to both therapeutic treatment and prophylactic or preventative measures. Preferably, it is a therapeutic treatment.

[0077] "Mammal" for purposes of treatment or therapy means any animal classified as a mammal, including humans, farm animals, and zoo, sport, or pet animals (dogs, horses, cats, cows, etc.). Preferably, the mammal is a human. Unless indicated otherwise, the terms "subject," "patient," and the like include mammals, including humans.

[0078] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by abnormal cell growth.

[0079] The term "nucleic acid" or "oligonucleotide," or grammatical equivalents, as used herein, refers to at least two nucleotides covalently linked together. The nucleic acids of the present invention are preferably single-stranded or double-stranded and generally contain phosphodiester bonds.

[0080] Amino acid sequence "variants" (or mutants) of antibodies are prepared by introducing appropriate nucleotide changes into the antibody DNA or by nucleotide synthesis. However, such modifications can only be made to a very limited extent, e.g., as described herein. For example, modifications do not change the antibody characteristics described above (e.g., IgG isotype and antigen binding), but may improve recombinant production yield, protein stability, or facilitate purification.

[0081] A "variant" of a molecule is a sequence substantially similar to that of the naturally occurring molecule. For nucleotide sequences, variants include sequences that, due to the degeneracy of the genetic code, encode the same amino acid sequence of the naturally occurring protein. Such naturally occurring allelic variants can be identified using well-known techniques of molecular biology, such as polymerase chain reaction (PCR) and hybridization techniques. Variant nucleotide sequences include those that encode polypeptides with amino acid substitutions, as well as nucleotide sequences of synthetic origin, such as those that encode naturally occurring proteins, generated, for example, by the use of site-directed mutagenesis. Generally, the nucleotide sequence variants of the present invention will, at least in one embodiment, be 40%, 50%, 60%, up to 70%, such as 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, up to 79%, typically at least 80%, such as 81%-84%, at least 85%, such as 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the naturally occurring (endogenous) nucleotide sequence.

[0082] The term "inhibit" refers to a decrease in an activity, response, condition, disease, or other biological parameter. Non-limiting examples can include a complete loss of activity, response, condition, or disease. Also included can be, for example, a 10% decrease in an activity, response, condition, or disease as compared to native or control levels. Thus, a decrease can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 100% decrease as compared to native or control levels, or any amount in between.

[0083] Compositions and pharmaceutical compositions

[0084] Another object of the present invention is a composition or pharmaceutical composition comprising at least one Hz monoclonal antibody or antigen-binding fragment thereof as disclosed and presented herein. The composition may further comprise a vehicle or diluent, particularly a vehicle or diluent suitable for the intended use of the antibody. When the composition is a pharmaceutical composition, a pharma- ceutically acceptable carrier, diluent, or

[0085] The pharmaceutical composition may comprise (i) at least one humanized anti-βig-h3 monoclonal antibody or antigen-binding fragment thereof according to the present invention, and (ii) at least one additional anti-tumor agent, such as an antibody directed to another target, and / or a chemotherapeutic agent, such as a small molecule. Both active ingredients may be present in the same composition. Alternatively, at least two of these active ingredients are separated, for example, into separate vials or compositions. In one aspect, a composition comprises at least two active ingredients as described herein for use in treating cancer and / or for modifying immunity, and is administered simultaneously, separately or sequentially to a mammal, including a human.

[0086] The additional active ingredient may be, inter alia, doxorubicin, gemcitabine, camptothecin, paclitaxel. The additional active ingredient may also be another antibody. The other antibody may be selected from the group consisting of another cancer marker or receptor, another antigen expressed on the surface of immune competent cells, an immune checkpoint, and combinations thereof.

[0087] Non-limiting examples of pharma- ceutically acceptable bases or excipients that can be used in these compositions include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (such as human serum albumin), buffer substances (such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts, or electrolytes (such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulosic substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and wool fat).

[0088] The pharmaceutical compositions of the present invention can be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, bucally, vaginally, or through an implanted reservoir. Utilized herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injection or infusion techniques. Sterile injectable forms of the compositions of the present invention can be aqueous or oleaginous suspensions. These suspensions can be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. Sterile injectable preparations can also be injectable solutions or suspensions in non-toxic, parenterally administrable diluents or solvents, such as solutions in 1,3-butanediol. Acceptable vehicles and solvents that can be used include water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are commonly used as solvents or suspension media. For this purpose, any bland, fixed oil can be used, including synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives, are useful in the preparation of injectables, as are natural pharma- ceutical acceptable oils, such as olive oil or castor oil, especially their polyoxyethylated versions. These oil solutions or suspensions can also contain long-chain alcohol diluents or dispersants, such as carboxymethylcellulose or similar dispersants commonly used in the preparation of pharma- ceutical acceptable dosage forms, including emulsions and suspensions. Other commonly used surfactants, such as Tween®, Span, and other emulsifiers or bioavailability enhancers utilized in the manufacture of pharma- ceutical acceptable solid, liquid, or other dosage forms, can also be used for formulation purposes.

[0089] The pharmaceutical compositions of the present invention can be administered orally in any orally acceptable dosage form, including, but not limited to, capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral use, commonly used bases include lactose and cornstarch. Lubricants, such as magnesium stearate, are also typically added. Diluents useful for oral administration in capsule form include, for example, lactose. When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring, or coloring agents can also be added. Alternatively, the compositions of the present invention can be administered in the form of suppositories for rectal administration. These can be prepared by mixing the drug with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature, and thus melts in the rectum to release the drug. Such materials include cocoa butter, beeswax, and polyethylene glycols. The compositions of the present invention can also be administered topically, especially when the target of treatment includes areas or organs that are easily accessible by topical application, including diseases of the eye, skin, or lower intestinal tract. Suitable topical formulations are easily prepared for each of these areas or organs. For topical application, the composition can be formulated into a suitable ointment containing the active ingredient suspended or dissolved in one or more bases. Non-limiting examples of bases for topical administration of the compounds of the present invention include mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compounds, emulsifying wax, and water. Alternatively, the composition can be formulated into a suitable lotion or cream containing the active ingredient suspended or dissolved in one or more pharma-ceutically acceptable bases. Non-limiting examples of suitable bases include mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol, and water. Topical application for the lower intestinal tract can be achieved in a rectal suppository formulation (see above) or a suitable enema formulation. Patches can also be used. The compositions of the present invention can also be administered by nasal aerosol or inhalation.Such compositions are prepared according to techniques well known in the art of pharmaceutical formulation and may be prepared as solutions in saline using benzyl alcohol or other suitable preservatives, absorption enhancers to enhance bioavailability, fluorocarbons, and / or other common solubilizing or dispersing agents.

[0090] For example, the antibody present in the pharmaceutical composition of the invention can be supplied at a concentration of 10 mg / mL in either 100 mg (10 mL) or 500 mg (50 mL) single-use vials. For intravenous administration, the product can be formulated in 9.0 mg / mL sodium chloride, 7.35 mg / mL sodium citrate dihydrate, 0.7 mg / mL polysorbate 80, and sterile water for injection. The pH can be adjusted to 6.5.

[0091] Pharmaceutical compositions of the invention for injection (e.g., intramuscular, intravenous) may be prepared to contain a pharma- ceutically acceptable base, diluent, or excipient, such as sterile buffered water (e.g., 1 ml for intramuscular injection), and from about 1 ng to about 100 mg, for example, from about 50 ng to about 30 mg, or more preferably from about 5 mg to about 25 mg, of an antibody of the invention.

[0092] In certain embodiments, liposomes and / or nanoparticles are contemplated for use in introducing antibodies into host cells. The formation and use of liposomes and / or nanoparticles are known to those skilled in the art. Nanocapsules are generally capable of accommodating compounds in a stable and reproducible manner. To avoid side effects due to intracellular polymer overload, such ultrafine particles (approximately 0.1 μm in size) are generally designed with polymers that are biodegradable. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these criteria are contemplated for use in the present invention and are easy to fabricate. Liposomes are formed from phospholipids dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also called multilamellar vesicles (MLVs)). MLVs generally have diameters between 25 nm and 4 μm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200-500 angstroms and containing aqueous solution in their cores. The physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations.

[0093] For use, methods of use (e.g. therapeutic treatment), use for manufacturing

[0094] Except where inappropriate, any feature disclosed herein applies to the different purposes of the invention (such as "for use," "method of use," or "therapeutic," "use for the manufacture of a medicament," etc.). In one embodiment, the patient or subject is a mammal, preferably a human.

[0095] Another object of the present invention is a humanized anti-βig-h3 monoclonal antibody or an antigen-binding fragment thereof, or a pharmaceutical composition disclosed herein for use as a medicine.

[0096] In one aspect, the invention relates to such a humanized anti-βig-h3 monoclonal antibody or antigen-binding fragment thereof, or a composition containing same, for use in (i) the treatment of solid cancers, or (2i) as an immunomodulatory composition. In particular, the immunomodulatory effect may be helpful in the treatment or during the treatment of cancer. The immunomodulation may include the restoration or activation of the activity of CD8+ T cells.

[0097] In another aspect, the present invention relates to such a humanized anti-βig-h3 monoclonal antibody or antigen-binding fragment thereof, or a composition comprising same, for use in reducing stromal stiffness, in particular by this function allowing other anti-tumor agents (antibodies, monoclonal antibodies, etc.) to gain access to the tumor.

[0098] The invention therefore generally relates to such humanized anti-βig-h3 monoclonal antibodies or antigen-binding fragments thereof, or compositions containing same, for use in the treatment of solid tumors.

[0099] In one embodiment, the solid tumor is one in which βig-h3 is expressed in the stroma.

[0100] In a preferred embodiment, the solid tumor may be or is selected from the list consisting of breast cancer, uterine / cervical cancer, esophageal cancer, pancreatic cancer, colon cancer, colorectal cancer, renal cancer, ovarian cancer, prostate cancer, head and neck cancer, non-small cell lung cancer, gastric cancer, tumors of origin (i.e. fibrosarcoma and rhabdomyosarcoma), tumors of the central and peripheral nervous system (i.e. astrocytoma, neuroblastoma, glioma, glioblastoma included) thyroid cancer. Preferably, the solid tumor is pancreatic cancer, esophageal squamous cell carcinoma, gastric and hepatic cancer, colon cancer, or melanoma. In a preferred embodiment, the solid tumor is pancreatic cancer. More preferably, the pancreatic cancer is pancreatic ductal adenocarcinoma.

[0101] The present invention also relates to a method of treating a solid tumor, comprising administering to a patient in need thereof a sufficient amount of such an antibody or antigen-binding fragment thereof, or a pharmaceutical composition comprising same.

[0102] The present invention also relates to a method of immunomodulation, comprising administering to a patient in need thereof a sufficient amount of a humanized anti-βig-h3 monoclonal antibody or antigen-binding fragment thereof, or such a medicament or immunomodulatory composition, said antibody or fragment being capable of helping restore or activate the activity of CD8+ T cells.

[0103] As used herein, the terms "treatment" and "treating" refer to curative or disease-modifying treatment, including treatment of patients having or diagnosed with cancer, particularly cancers whose stroma express βig-h3, including inhibition of clinical recurrence. Treatment can relate to a subject with cancer and is aimed at curing, delaying the onset, reducing the severity, or ameliorating one or more symptoms of said cancer, or at prolonging the subject's survival beyond that expected without such treatment.

[0104] The disclosed antibodies or antigen-binding fragments thereof can be administered as therapeutic agents to a subject (particularly a human) in an amount ranging from about 0.001 mg to about 100 mg, about 0.01 mg to about 50 mg, about 0.1 mg to about 40 mg, about 0.5 mg to about 30 mg, about 0.01 mg to about 10 mg, about 0.1 mg to about 10 mg, or about 0.5 mg to about 25 mg per kg of subject body weight per day, or about 0.5 to about 10, 5, 3, or 2 mg, one or more times per day to achieve the desired therapeutic effect. The desired dose can be delivered three times per day, twice per day, once per day, every 2 days, every 3 days, every week, every 2 weeks, every 3 weeks, or every 4 weeks. In certain embodiments, the desired dose can be delivered using multiple administrations (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more administrations).

[0105] Administration can be, for example, intravenous, intramuscular, intraperitoneal, or subcutaneous, e.g., proximal to the target site. The dosing regimen in the above treatment methods and applications is adjusted to provide the optimum desired response (e.g., therapeutic response). For example, a single bolus can be administered, several divided doses can be administered over time, or the dose can be proportionally reduced or increased as the therapeutic situation dictates. In some embodiments, the effectiveness of the treatment is monitored, e.g., at predetermined time points during the treatment. In some embodiments, the effectiveness can be monitored by visualization of the diseased area or by other diagnostic methods further described herein (e.g., by performing one or more PET-CT scans using the labeled antibodies or antigen-binding fragments thereof of the invention). If desired, an effective daily dose of the pharmaceutical composition can be administered as two, three, four, five, six, or more subdoses administered separately at appropriate intervals throughout the day, optionally in unit dosage form. In some embodiments, the monoclonal antibodies of the invention are administered by slow continuous infusion over an extended period of time (e.g., more than 24 hours) to minimize any undesired side effects. Effective doses of antibodies of the invention may also be administered using weekly, biweekly, or triweekly dosing periods, which may be limited to, for example, 8 weeks, 12 weeks, or until clinical progression is established.As a non-limiting example, treatment according to the invention can be performed by administering to a subject (particularly a human) a daily dose of an antibody or antigen-binding fragment thereof of the invention in an amount of about 0.1 to 100 mg / kg per day (e.g., 0.2, 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90, or 100 mg / kg) for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90, or 100 mg / kg per day) for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, at least one of 3, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 days, or at least one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 weeks, or any combination thereof, using a single dose or using divided doses every 24, 12, 8, 6, 4, or 2 hours, or using any combination thereof.

[0106] combination

[0107] The present invention also provides therapeutic applications or methods of treatment in which the antibody or antigen-binding fragment thereof of the present invention is combined with at least one additional therapeutic agent, for example, for treating cancer. Such administration can be simultaneous, separate, or sequential; that is, treatment with the two active ingredients can be at the same time (e.g., simultaneously or in tandem), or at different times (e.g., consecutively or sequentially), or a combination thereof. The additional therapeutic agent is typically related to the disorder to be treated. Representative therapeutic agents include other anti-cancer antibodies, cytotoxic agents, chemotherapeutic agents, anti-angiogenic agents, anti-cancer immunogens, cell cycle control / apoptosis regulators, hormone regulators, and other agents described below.

[0108] In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention are used in combination with chemotherapeutic agents or antibodies, particularly monoclonal antibodies (such as ICIs) that specifically target tumor antigens, receptors, or ligands. The term "therapeutic agent" refers to a compound that is effective in inhibiting tumor growth.

[0109] Thus, according to the present invention, there is provided a method for the simultaneous or sequential use in the treatment of solid tumors, comprising: i. a humanized antibody or antigen fragment thereof disclosed herein; ii. Therapeutic agents disclosed herein for treating cancer A combination of is provided.

[0110] In particular, the present invention provides a method for the simultaneous or sequential use in the treatment of solid tumors, comprising administering to said patients: i. a humanized antibody or antigen fragment thereof disclosed herein; ii. Immune checkpoint inhibitors (ICIs) A combination of is provided.

[0111] Typically, the checkpoint blockade cancer immunotherapeutic agent is an antibody. In some embodiments, the checkpoint blockade cancer immunotherapeutic agent is an antibody selected from the group consisting of anti-CTLA4 antibody, anti-PDl antibody, anti-PDLl antibody, anti-PDL2 antibody, anti-TIM-3 antibody, anti-LAG3 antibody, anti-IDOl antibody, anti-TIGIT antibody, anti-B7H3 antibody, anti-B7H4 antibody, anti-BTLA antibody, and anti-B7H6 antibody. These antibodies are preferably monoclonal antibodies or antigen-binding fragments thereof. The antibody can in particular be a PD-1 blocking antibody (pembrolizumab, nivolumab, atezolizumab, avelumab, or durvalumab), or a CTLA-4 blocking antibody (ipilimumab). This combination is based on the disclosure of WO2020079164, which describes the combination of ch18B3 and ICI. This document is incorporated herein by reference.

[0112] The antibody or antigen-binding fragment of the present invention may be used in combination with existing chemotherapy to target βig-h3 protein, which may be more effective at killing tumor cells than chemotherapy alone.Non-limiting examples include cisplatin, taxol, etoposide, mitoxantrone, actinomycin D, camptothecin, methotrexate, gemcitabine, mitomycin, dacarbazine, 5-fluorouracil, doxorubicin, and daunomycin.

[0113] The antibodies or antigen-binding fragments thereof of the present invention can be used in combination with immune checkpoint inhibitors (such as anti-PD1, anti-PD-L1, or CTLA4 antibodies) as additional anti-cancer agents.

[0114] In one method of the invention, the βig-h3 binding antibody or fragment is administered to the patient prior to administration of a second anti-cancer agent.

[0115] Generation of antibodies

[0116] The antibodies and antigen-binding fragments thereof of the present invention are produced by utilizing any technique known in the art, including, but not limited to, any chemical, biological, genetic, or enzymatic technique, either alone or in combination. Typically, once the amino acid sequence of a desired sequence is known, one of skill in the art can readily produce said antibody by standard techniques for polypeptide production. For example, antibodies can be synthesized using well-known solid-phase techniques, preferably using a commercially available peptide synthesizer (e.g., manufactured by Applied Biosystems, Foster City, Calif.), following the manufacturer's instructions. Alternatively, antibodies of the present invention can be synthesized by recombinant DNA techniques well-known in the art. For example, antibodies can be obtained as DNA expression products after incorporating DNA sequences encoding the antibodies into an expression vector and introducing such vector into a suitable eukaryotic or prokaryotic host that will express the desired antibodies, from which the antibodies can later be isolated using well-known techniques.

[0117] Mammalian cells are the preferred host for generating therapeutic antibodies due to their ability to glycosylate proteins in a form optimal for human application. Bacteria glycosylate proteins very rarely, and other similar types of common hosts (such as yeast, filamentous fungi, insect, and plant cells) produce glycosylation patterns associated with rapid clearance from the bloodstream. Among mammalian cells, Chinese hamster ovary (CHO) cells are the most commonly used. In addition to providing the appropriate glycosylation pattern, these cells allow for the consistent generation of genetically stable and highly productive clonal cell lines. These cells can be cultured at high densities in serum-free media in simple bioreactors, allowing for the development of safe and reproducible bioprocesses. Other commonly used animal cells include baby hamster kidney (BHK) cells, NSO mouse myeloma cells, and SP2 / 0 mouse myeloma cells.

[0118] In one embodiment, the antibody according to the invention is produced or expressed in mammalian cells, preferably wild-type mammalian cells, preferably of rodent origin, in particular CHO cells.

[0119] Modifications and changes can be made in the structure of the antibodies of the present invention and still obtain molecules with similar characteristics. For example, certain amino acids in the sequence can be substituted with other amino acids without significant loss of activity. Since the interactive ability and properties of an antibody define its biological functional activity, certain amino acid sequence substitutions can be made in the antibody sequence (or, of course, the underlying DNA coding sequence) to obtain antibodies with similar properties. When making such changes, the hydropathic index of the amino acids can be taken into consideration. The importance of the amino acid hydropathic index in conferring interactive biological function to an antibody is generally understood in the art. It is known that certain amino acids can be substituted with other amino acids with similar hydropathic indexes or scores and still obtain antibodies with similar biological activity. Each amino acid is assigned a hydropathic index based on its hydrophobicity and charge characteristics.

[0120] It is believed that the relative hydropathic characteristics of amino acids determine the secondary structure of the resulting antibody, which in turn determines the interaction of the antibody with other molecules (e.g., enzymes, substrates, receptors, antibodies, antigens, etc.). It is known in the art that one amino acid can be substituted with another having a similar hydropathic index and still obtain a biologically equivalent polypeptide. Among such changes, substitution of amino acids whose hydropathic index is within ±2 is preferred, those within ±1 are particularly preferred, and those within +0.5 are even more particularly preferred.

[0121] Substitution of like amino acids can also be made on the basis of hydropathicity, particularly where biologically functional equivalent peptides or polypeptides are thereby created and contemplated for use in immunological embodiments. U.S. Patent No. 4,554,101, which is incorporated herein by reference and which may be consulted by those of skill in the art, states that the maximum local average hydrophilicity of a polypeptide, as governed by the hydrophilicity of adjacent amino acids, correlates with the immunogenicity and antigenicity of the polypeptide (i.e., the biological properties of the polypeptide).

[0122] As detailed in U.S. Patent No. 4,554,101, the following hydrophilicity values ​​have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid (+3.0±1); glutamic acid (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); proline (-0.5±1); threonine (-0.4); alanine (-0.5); histidine (-0.5); cysteine ​​(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). It is understood that substitution of one amino acid with another having a similar hydrophilicity value may result in a biologically equivalent, and in particular an immunologically equivalent, polypeptide. In such changes, substitution of amino acids whose hydrophilicity values ​​are within ±2 are preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

[0123] As outlined above, amino acid substitutions are therefore generally based on the relative similarity of the amino acid side-chain substituents (e.g., their hydrophobicity, hydrophilicity, charge, size, etc.). The amino acid substitutions can be differently selected or chosen. Possible substitutions are described in WO99 / 51642, WO2007024249, and WO2007106707.

[0124] Nucleic acids and vectors

[0125] The isolated nucleotide sequences disclosed and provided herein are also objects of the present invention. Thus, the present invention also relates to isolated nucleotide sequences selected from the group consisting of nucleotide sequences SEQ ID NO: 21, 22, 23, and 24, more particularly a combination or set of two nucleotide sequences, separated or linked together: SEQ ID NO: 21 and 23, 21 and 24, 22 and 23, 22 and 24. The nucleotide sequence encoding H-330V1.2 is as shown in SEQ ID NO: 29, and when the mutated version C102S is desired, it can be used in place of SEQ ID NO: 21 to generate monoclonal antibodies.

[0126] As mentioned above, methods for producing antibodies are known to those skilled in the art. Mammalian cells, preferably rodent cells (such as CHO cells), preferably wild-type cells, are transfected with one or several expression vectors. The cells are preferably simultaneously transfected with an expression vector for the light chain and an expression vector for the heavy chain. The transfection of cells is also known to those skilled in the art. Non-limiting examples of transfection that can be carried out include calcium phosphate precipitation, DEAE-dextran mediated transfection, electroporation, magnetofection, nucleofection (AMAXA Gmbh, Germany), transfection through liposomes (e.g. using dreamfect®, lipofectin® or lipofectamine® technology) or microinjection, which are standard transfection procedures well known to those skilled in the art.

[0127] Expression vectors are known. Non-limiting examples of vectors that can be used include pcDNA3.3, pOptiVEC, pFUSE, pMCMVHE, pMONO, pSPORT1, pcDV1, pcDNA3, pcDNA1, pRc / CMV, pSEC. A single expression vector or several expression vectors expressing different parts of the antibody can be used.

[0128] The present invention also relates to an expression vector encoding a heavy chain of an Hz antibody of the invention, an expression vector encoding a light chain of an Hz antibody of the invention, or an expression vector encoding the heavy and light chains of such an Hz antibody.

[0129] Another object of the present invention is a host cell containing a vector or a group of vectors of the present invention.The host cell can be a mammalian cell, preferably a rodent cell, more preferably a CHO cell.It is even more preferred that the host cell is a wild-type mammalian cell, preferably a wild-type rodent cell, most preferably a wild-type CHO cell.

[0130] The skilled artisan is well trained on how to use such vector(s) and cells (such as CHO cells) to produce an antibody according to the invention.

[0131] Methods of measurement used herein (see also the Examples for completeness):

[0132] Affinity and dissociation rate [Table 2]

[0133] DSC

[0134] Differential scanning calorimetry (DSC) is an analytical technique used to directly characterize the stability of proteins or other biomolecules in their native form by measuring the heat changes associated with the thermal denaturation of the molecule as it is heated at a constant rate.

[0135] DSC protocol utilized herein: ·- Differential scanning calorimetry experiments were carried out using a MicrocalTM VP-capillary DSC system. ·- Samples were stored at -20°C. After thawing, all samples were centrifuged (20.000g, 5 min, 4°C) and, when necessary, diluted in PBS to a concentration of 1 mg / mL. Prior to DSC analysis, the protein content of the samples was quantified with a Nanodrop ND-1000 spectrophotometer (s / n: 4847) with the IgG analysis program. - Pre-equilibration time was 3 min, and subsequent thermograms were acquired between 20 and 110 °C using a scan rate of 60 °C / h, a filtering period of 25 s, and medium feedback. Prior to sample analysis, five buffer / buffer scans were measured to stabilize the instrument, and one buffer / buffer scan was performed between each protein / buffer scan. ·- Data were fitted to a non-two-state unfolding model with subtracted adjusted baselines before and after the transition. Calorimetric enthalpies (ΔH) are determined as the area under the transition peaks, whereas van't Hoff enthalpies (ΔHv) are determined from the model used. The solid red line represents the measured data, the solid black line represents the best fit for the model used, and the grey line represents the contribution from a single unfolding unit to the overall protein unfolding.

[0136] General considerations for DSC: - Tm or denaturation / melting temperature is the midpoint of the unfolding transition at which the concentrations of unfolded and folded species are equal. Tm is a parameter that describes the susceptibility of a protein to thermal denaturation and is therefore related to the stability of the protein. The higher the Tm, the more stable the protein. ·- Tonset is the temperature at which the unfolding transition begins. This parameter is usually 5-10°C lower than Tm. It is also a parameter that indicates the stability of a protein, but is related to its resistance to thermal denaturation. ·- T1 / 2 is the width of the transition at half the peak height. It represents the width of the transition, typical values ​​are between 1 and 15 °C. This parameter correlates with the compactness of the protein packing, with broader transitions corresponding to less compact proteins. ·- ΔH is the calorimetric enthalpy of unfolding, reflecting the disruption of intramolecular interactions of a protein (i.e., the disruption of intra- and inter-domain interactions). This process is endothermic, giving a positive enthalpy value. ·- Total area is the total enthalpy of unfolding (total area under the thermogram) and reflects the thermodynamic stability. - Monoclonal antibodies typically exhibit complex, multi-domain thermograms. The largest and most prominent domain peak is the Fab of the antibody. CH2 and / or CH3 domains are also commonly found. The relative positions of the different domains and TMs depend on the specific monoclonal antibody and may vary depending on subclass and engineering.

[0137] Transient transfection in CHO cells

[0138] Humanized "V1 version" antibodies were produced in CHO cells. CHO DG44 cells were maintained in ventilated Erlenmeyer flasks (Corning) at 37°C and 5% CO2 on an orbital shaker. The day before transfection, cells were passaged at a defined density (according to MO.CEL.120). On the day of transfection (day 0), cells were mixed with transfection reagent and plasmid DNA to generate pilot lots (30 ml).

[0139] The invention will now be described by way of non-limiting examples and with reference to the drawings, in which: EXAMPLES

[0140] Example 1: Epitope Mapping

[0141] Epitope mapping studies showed that this antibody targets the fourth domain of FAS1 (linear epitope ALPPRERSRL) (AA residues 549-558) of the βig-h3 protein. See Figure 1. It was possible to further restrict the epitope to LPPRERSR.

[0142] Example 2:

[0143] The proof-of-concept (PoC) phase aims to demonstrate that the 18B3 mAb (Bae et al., 2014 Acta Physiol 2014, 212, 306-315), directed against the βig-h3 protein, efficiently and specifically depletes the βig-h3 protein and thus (i) inhibits CD8 + "We demonstrate that IL-16 can play a key role in cancer therapy by (i) increasing the cytotoxic activity of T cells and (ii) decreasing stromal stiffness, thereby restoring immune system access to the tumor, ultimately leading to significant tumor regression and survival in mice."

[0144] To that end, numerous experiments were performed both in vitro (in relevant mouse models) and in vivo. It should be noted that the majority of experiments were performed in vivo that provide evidence that anti-βig-h3 therapeutic mAbs may be effective, alone or in combination, in addressing PDAC and potentially other cancers.

[0145] In brief, the conclusions of this PoC phase are:

[0146] 1. Targeted depletion of βig-h3 protein by 18B3 mAb in PDAC models allows: a. limiting tumor growth; b.CD8 + Restoring the cytotoxic activity of T lymphocytes; C. reducing interstitial stiffness;

[0147] This was demonstrated in both in vitro and in vivo assays specially developed by a scientific team with this purpose in mind.

[0148] 2. Experiments performed with two different mouse strains developing pancreatic tumors showed that there was significant tumor shrinkage and increased survival in populations treated with 18B3 by subcutaneous injection twice a week for three weeks.

[0149] 3. Several experiments performed with higher doses of 18B3 demonstrated that increasing concentrations of the mAb further reduced tumor growth and increased mouse survival.

[0150] 4. We demonstrated that 18B3 mAb treatment significantly reduced tumor growth in mouse HSG ovarian and bladder cancers, respectively, suggesting that 18B3 mAb may be a potential complement or alternative to anti-PD1 in anti-PD1-resistant bladder cancer.

[0151] The overall conclusion of this comprehensive set of data is that the concept of using mAbs that deplete βig-h3 protein as a therapy is validated.

[0152] Example 3: Chimerization and humanization of 18B3 murine mAb

[0153] 1. Method

[0154] The project was carried out in four stages: Phase 1: Sequencing of mouse 18B3 mAb Step 2: Chimerization of mouse 18B3 mAb Step 3: Design of humanized variants by CDR grafting (antibody reshaping) Phase 4: Creation and analytical testing of humanized variants

[0155] Phase 1: Sequencing of mouse 18B3 mAb

[0156] The VH and VL domains of mouse 18B3 (isotype IgG1 / k) were sequenced from hybridoma cells using cDNA sequencing.

[0157] For this purpose, RNA was extracted from the hybridoma cells. The corresponding DNA strand was synthesized by high-fidelity RT-PCR, followed by second strand synthesis to obtain double-stranded cDNA. The cDNA was then sequenced and translated into a protein sequence. VH sequence: SEQ ID NO: 17 VL sequence: SEQ ID NO: 18

[0158] Step 2: Chimerization of mouse 18B3 mAb

[0159] Chimerization consists in replacing the constant domains of the murine 18B3 mAb by human sequences. The sequences coding for the variable domains of the heavy chain (VH SEQ ID NO: 19) and the variable domains of the light chain (VL SEQ ID NO: 20) were optimized for expression in mammalian cells and synthesized. The corresponding synthetic genes were then cloned into a vector system containing the human constant regions of the IgG1 heavy chain (SEQ ID NO: 25) and the kappa light chain (SEQ ID NO: 26). The vectors, once verified by sequencing, are amplified to prepare low-endotoxin plasmid DNA, which is further verified by sequencing.

[0160] The chimeric mAb was then produced by transient expression of the plasmid in CHO mammalian cells and subsequently purified: CHO DG44 cells were maintained in ventilated Erlenmeyer flasks (Corning) at 37°C and 5% CO2 on an orbital shaker. The day before transfection, cells were passaged at a defined density. On the day of transfection (day 0), DNA plasmids encoding the light and heavy chains of the 18B3 chimeric antibody were added to the cell suspension and mixed with the transfection reagent to generate pilot lots.

[0161] The supernatant was purified by protein A affinity chromatography. After dialysis in PBS and sterile filtration (0.22 μm), the total protein concentration was determined by spectrophotometric reading at 280 nm. The purified chimeric mAb was then stored at temperatures below -20°C.

[0162] The integrity of the chimeric mAb was examined by SDS-PAGE. The affinity of the chimera for the ligand was compared to that of the parental murine 18B3 mAb by ELISA.

[0163] For ELISA:

[0164] Antigen (recombinant human βIG-H3: rhβIG-H3, R&D systems, catalog number 3409-BG, lot NDM061911A) was coated at 1 μg / ml overnight at 4°C.

[0165] Antigens were removed and nonspecific sites were blocked with PBS-milk at 2.5% for 1 h at room temperature.

[0166] The mAbs to be tested (anti-hβIG-h3 18B3 parental (mouse) or chimeric) were added at 10 μg / ml, diluted 10-fold across seven wells, and incubated for 2 hours at room temperature.

[0167] After removing and washing the plates, secondary antibodies were added (HRP-labeled anti-mouse IgG for mouse 18B3 (parental) and HRP-labeled anti-human IgG (Fc specific) for chimeric 18B3) and incubated for 2 hours at room temperature.

[0168] Upon addition of a colorless substrate (TMB: 3,3',5,5'-tetramethylbenzidine), the HRP reacts to produce a blue color, and the spectrophotometric signal is proportional to the amount of mAb bound to the antigen.

[0169] The reaction was stopped by adding sulfuric acid, which turned the TMB yellow, and the amount of mAb was assessed by spectrophotometry (optical density) at 450 nm.

[0170] Chimerization was successful, resulting in very similar biophysical characteristics compared to the parental murine antibody.

[0171] Step 3: Design of humanized 18B3 variants by CDR grafting (antibody engineering)

[0172] The aim of this step is to obtain several variants of chimeric 18B3 that are further humanized to reduce the immunogenicity and increase the half-life of the mAb in humans. It is considered that the % of humanity should be greater than 85%.

[0173] Humanization was performed using CDR grafting technology.

[0174] The humanization strategy involves a combination of techniques, namely: Primary sequence analysis and alignment. - 3D modeling, · Selection of the best human germline Based on.

[0175] Indeed, the combination of structural (3D) models and pure sequence analysis allows the identification of actual paratope-facing and non-paratope residues in potential CDR regions. In addition, the structural models can drive the selection for backmutations in light of the selected germline scaffold used, allowing a faster humanization process.

[0176] Note that the Kabat numbering system was used to identify residues.

[0177] The first step is the selection of germline human VH and VL sequences as close as possible to the mouse 18B3 mAb. To design the CDR-grafted version of the anti-HuβIG-H3 18B3 murine VH, three human germlines (IGHV3-11*01, IGHV3-30*01, and IGHV1-69*08) were selected (see Table 2). The human germline IGHV3-11*01 was chosen because of its high sequence identity with mAb 18B3-VH across the entire V gene; 76.5% (75 residues matched out of a total of 98). IGHV3-30*01 and IGHV1-69*08 were chosen despite the fact that they have less sequence identity (72.4% and 55%, respectively) because they are widely used germline sequences for human antibody generation (according to the IMGT / GeneFrequency database) and have other interesting features, offering the possibility of grafting CDRs in different molecular environments, especially for IGHV1-69*08. For H-CDR3 and the N-terminal part of framework 4, the human germline IGHJ6*01 (J-GENE) was selected as the closest candidate. · Three human germlines (IGKV4-1*01, IGKV2-28*01, and IGKV3-15*01) appeared to be the best choices to design the CDR-grafted version of anti-HuβIG-H3 18B3 mouse VLκ, because the percent sequence identity of the human framework acceptor regions for all three human germlines was 82.2% for IGKV4-1*01, 67.3% for IGKV2-28*01, and 64.4% for IGKV3-15*01. For the L-CDR3 and the N-terminal part of framework 4, the human germline IGKJ2*01 (J-GENE) was selected as the closest candidate.

[0178] · The second step is to graft the CDRs of the 18B3 mouse mAb onto the human germline of the selected VH and VL without introducing any mutations in the sequence: this is called the V0 version of the humanized variant.

[0179] The third step is to optimize the obtained sequences to (i) maintain the functionality of the humanized variant and (ii) increase the % of humanness if possible, resulting in humanized variant versions V1 to V3. The optimization was performed as follows: · Several amino acid residues in the framework regions (Fr) of selected human germline variable regions were inverted (backmutated) to the corresponding murine amino acid residues. Based on the information gathered on the structure of immunoglobulin variable regions and the guidance of the molecular model of mAb 18B3 (VH and VL), several of these residues in Fr were identified as potentially having key roles in maintaining the conformation of the CDRs or playing a role in the interface between the heavy and light chain variable regions. ·Also, VL and VH are two domains that interact without forming a covalent bond. The interaction between these two domains is maintained through hydrogen and electrostatic bonds. The residues involved in this interaction must also be maintained, otherwise the paratope may change and the affinity of the antibody may change. Therefore, they were maintained in the humanized version V1 and the V1.2 mutant version C102S.

[0180] The % human results obtained are summarized in Table 2 below: [Table 3]

[0181] Phase 4: Creating and testing humanized variants

[0182] The aim of this step is to generate selected humanized variants by combining the 3 selected VHs with the 3 selected VLs, thus resulting in 9 different variants.

[0183] Construction and purification were performed exactly as described for the chimeras, using VH and VL nucleotide sequences SEQ ID NOs: 21-24 where appropriate.

[0184] Analytical testing of the nine generated humanized variants was performed using several methods: % of humanization achieved by sequence alignment and germline selection Productivity was assessed by the level of transient expression in mammalian cells. Antibody affinity was assessed by ELISA as described above (step 2 chimerization). Thermal stability assessed by Differential Scanning Calorimetry (DSC): measured using a MicrocalTM VP-Capillary DSC system as described in the method of measurement above. Purity (aggregation) was checked by High-Performance Size Exclusion Chromatography (HP-SEC): Shimadzu Prominence HPLC with the following components: CBM-20A system controller, SPD-M20A photodiode array detector; column furnace CTO-20A; automatic sampling unit SIL-AC; pump LC-20AD, and degassing unit DGU-20A5. A Column Superdex 200 increase 5 / 150GL column from GE Healthcare was used. The column was previously equilibrated in PBS 1x at 0.25 mL / min with the column oven set at 30°C. All samples were centrifuged (20.000×g, 5 min, 4°C) and their protein content was quantified by a Nanodrop ND-1000 spectrophotometer with an IgG analysis program, followed by SEC analysis. When necessary, samples were diluted to a concentration of 1 mg / mL in PBS immediately before injection. The isocratic program was set to inject approximately 15 μg of each sample at 0.25 mL / min for 18 min. After SEC analysis, chromatograms at 280 nm were extracted from the raw data and analyzed by peak integration. The column was calibrated using a series of proteins from the molecular weight SEC calibration kit from GE Lifesciences under the same conditions and buffers used while analyzing the samples. The proteins used were aprotinin (6.500 Da), ribonuclease A (13.700 Da), carbonic anhydrase (29.000 Da), ovalbumin (44.000 Da), conalbumin (75.000 Da), and aldolase (158.000). The excluded volume of the column was determined using Blue Dextran. Calibration of the column was performed according to the gel filtration calibration kit instructions (GE Lifesciences).

[0185] Production and analytical testing of the nine variants showed that: All variants have excellent productivity, especially variants with heavy chains #330 and #311 (to a lesser extent); variants with heavy chain #169 have lower productivity than chimeric antibodies The affinity of the variants in the ELISA assay showed similar binding affinity compared to the chimera (and parent) in the ELISA; however, the variant using heavy chain #169 appears to be less favorable here as well. HP-SEC showed very high purity for all variants, with less than 2-3% potential aggregates. In DSC, the Tm of all variants was above 70°C, with the more stable variant being the variant with heavy chain #330 (higher and unexpected level of Tm than the chimeric antibody).

[0186] [Table 4] The reactivity of the four generated humanized variants against human βIg-h3 was relatively similar. No significant loss of reactivity against the target or degraded behavior regarding biophysical properties was observed: The variant with the closest germline gene, H311-V1, showed similar reactivity and stability characteristics to the chimeric mAb. The H330-V1 variants (especially the variants H330-V1 / L41-V1 and H330-V1 / L228-V1) showed unexpectedly high Fab Tm (potentially related to their reduced tendency to aggregate) and showed reactivity characteristics similar to those of chimeric mAbs. By analyzing the entire data set, the heavy chains containing H-311 or H-330 appeared to play a major role in binding to the target, whereas the light chains did not appear to play a major role in binding; therefore, H-311 or H-330 would be good choices to combine with different VL domains. · V1 candidates H-311 / L-41 (to utilize the closest germline genes for both VH and VL and their common properties) and H-330 / L-228 are preferred. The DSC of H330-V1.2 / L41-V1 and H330-V1.2 / L228-V1 is still above 80°C, and the stability properties are similar to those of the non-mutated versions (with H-330V1).

[0187] The % humanness obtained for the V1 candidates is very good.

[0188] The framework was slightly modified to increase its humanization while retaining AA residues known to be involved in the conformation ("orientation") of the CDRs.

[0189] In the CDRs, two mutations were introduced at positions 57 and 60 in CDR#2 of VH330 and VH311. 3D modeling showed that these AAs are not involved in the paratope and are “hidden” in the structure of the mAb.

[0190] [Table 5]

[0191] All six selected variants show a very high percentage of humanity, in the range of 89-97% as shown above in the specification, significantly reducing the risk of immunogenicity in humans in the future.

[0192] Example 4: ELISA

[0193] The analytical method is based on a direct ELISA test.

[0194] The antigen (recombinant human βig-h3 protein) is coated onto the surface of a 96-well plate. The mAb to be tested is then added.

[0195] A secondary anti-human mAb labeled with horseradish peroxidase (HRP) enzyme is then added.

[0196] Upon addition of a colorless substrate (TMB: 3,3',5,5'-tetramethylbenzidine), HRP produces a blue color signal that is proportional to the amount of mAb bound to the antigen.

[0197] The reaction is stopped by adding sulfuric acid and TMB, resulting in a yellow color.

[0198] The amount of mAb is assessed by spectrophotometry (optical density) at 450 nm.

[0199] The results obtained for chimeric 18B3 (considered as the reference mAb) and the four humanized lead variants are summarized in Figure 2. Statistical analysis (one-way ANOVA).

[0200] The results showed that the humanized variant H-311 / L-228 showed a lower affinity for the target compared to the chimera variant. The other three humanized variants did not show a statistically significant difference compared to the chimera, indicating that the humanization process did not change the affinity measured by ELISA. The three variants show very similar affinities by EC50 and statistical analysis, but the variant with heavy chain 330 consistently shows the best EC50 (and on average better than the chimera).

[0201] [Table 6]

[0202] Example 5: In vitro functional bioassay

[0203] The principle of this test is to test the efficacy of different mAbs directed against the anti-βig-h3 protein by depleting that protein, thereby inhibiting the cytotoxic CD8 + The aim of this study was to assess their ability to restore T cell activation and proliferation.

[0204] First, freshly extracted spleen CD8 + T cells are contacted with antigen-presenting cells (ie, bone marrow-derived cells bearing processed OVA peptide) to promote activation and proliferation.

[0205] Then, (CD8 + The rhβig-h3 protein (known to block T cell activation pathways) is added along with the mAb to be tested.

[0206] After 72 hours, CD8 + T cell activation and proliferation are quantitatively assessed by flow cytometry (FACs) analysis.

[0207] The efficacy of the mAbs is assessed statistically by comparing the levels of activation / proliferation with an isotype control (irrelevant) mAb.

[0208] Bone-derived bone marrow cells are obtained from the bones of C57BL6 wild-type mice and cultured in vitro for 5-6 days. They are then matured by incubating with LPS for 12 hours and activated as antigen-presenting cells by adding OVA peptide (SIINFEKEL). The OVA peptide is processed and presented on the surface of BMDCs (OVA-treated BMDCs).

[0209] In parallel, CD8 + T cells are extracted from the spleens and lymph nodes of OT1 mice by disruption and preparation of a single cell suspension.

[0210] OVA-bearing BMDCs and CD8 + T cells are combined with rh-βig-h3 protein and the mAb to be tested. They are then incubated at 37° C. for 72 hours to identify CD8 + Enables T cell activation and proliferation.

[0211] Next comes CD8 + T cell proliferation and activation were assessed at 4°C by FACS staining and analyzed with FlowJo software.

[0212] Statistical significance of proliferation is assessed through Student's t-test and one-way analysis of variance performed using GraphPad Prism software.

[0213] The results obtained for chimeric 18B3 (considered as the reference mAb), as well as the four humanized lead variants and the negative control, are summarized in Figure 3. Statistical analysis (one-way ANOVA).

[0214] The results obtained showed that all humanized variants have a statistically significant functionality against the target compared to the negative control. This functionality is statistically completely equivalent to the reference mAb (chimeric 18B3). As a result, the in vitro functional tests show that engineering the selected humanized variants did not alter their in vitro functionality against their specific targets. Although all these variants are very similar, the ranking shows that the variant with heavy chain H-330 gives the best results, with variant H-330 / L-41 being the most efficient.

[0215] Example 6: In vivo functional bioassay

[0216] The objective of this set of experiments was to demonstrate that co-administration of 18B3 mAb with PDAC-specific tumor cells limits tumor growth (as assessed by the number of tumor cells in the graft) and inhibits CD8 + The goal is to demonstrate that it is possible to restore T cell activation pathways.

[0217] The specific intent of this experiment is to show that the above effects are proportional to the amount of 18B3 administered.

[0218] PDAC tumor cells are obtained from isolated pancreases of 2.5 month old KIC mice and cultured in vitro.

[0219] KIC cells are embedded as clusters in a 1:1 mixture of Matrigel (Corning) and injected subcutaneously into the flank of normal C57BL6 mice with increasing doses of 18B3 mAb per mouse. Controls consist of an irrelevant isotype control IgG1 mAb administered at the highest dose of 18B3 evaluated. The same mouse population (n=8) is used for each dose evaluated.

[0220] Mice are then monitored for 10 days before being euthanized.

[0221] Tumor grafts are then weighed, measured, and single cell suspensions obtained by digestion with collagen buffer and processed for staining followed by flow cytometry.

[0222] The amount of tumor cells in the graft and CD8 + T cell proliferation and activation is then assessed by FACS staining at 4° C. and analyzed with FlowJo software.

[0223] The statistical significance of parameters is assessed through Student's t-test and one-way analysis of variance performed using GraphPad Prism software.

[0224] The results show that statistical differences are found between the humanized candidates and the chimeras and between the various humanized candidates. The humanized variant with heavy chain H-330 shows better results overall. The humanized variant H-330 / L-228 is the lead variant and shows values ​​comparable or even better than the chimeras. This variant also shows the best homogeneity (lowest variance-SEM) for all three parameters. The humanized variant H-330 / L-228 shows the best profile.

[0225] Example 7: Surface plasmon resonance (Biacore) - affinity, association and dissociation rates.

[0226] The assay was performed using a Biacore T200 instrument. It was carried out in two steps: The first step, which we call suitability testing, aimed to determine the optimal conditions for performing the assay: here, the best sensor chip and running buffer conditions were evaluated, resulting in the highest defined signal / noise ratio. The second step was the run itself, which was performed in triplicate to ensure reproducibility and robustness of the results.

[0227] For the first step, two sensors and different buffer conditions were evaluated using the parental 18B3 mAb and the chimeric 18B3 mAb as a reference, revealing that sensor chip C1 was optimal and that a buffer containing 300 mM NaCl and 0.25 mg / mL BSA reduced nonspecific binding and increased the signal.

[0228] As for the run itself, the protocol was as follows: 1. Reversible immobilization (covalent immobilization) of the test antibody with an anti-mouse or anti-human IgG secondary antibody. 2. Interaction analysis of antigen and captured antibody. 3. Regeneration: Complete removal of antibody and antigen from the surface S of the secondary antibody.

[0229] Further details are given in the Measurement Methods section.

[0230] The rate constants (k a , k d ) and the equilibrium dissociation constant (K D ). Experimental data were fitted to a 1:1 binding model. Shown are the mean ± SD of n = 3 independent experiments.

[0231] [Table 7]

[0232] The optimized settings for the SPR assay worked very well, providing highly accurate and reproducible results.

[0233] All mAbs have affinities (KD) in the subnanomolar range.

[0234] There are small overall differences in ka, kd, and KD for all samples: The association rate (ka) of the mouse antibody m18B3 is slightly slower than that of all other samples. The dissociation rate (kd) is surprisingly slower for the humanized (Hz) variant. The overall KD values ​​(affinity) were lowest for the chimeric antibody ch18B3 (0.2nM), with the parental mouse and fully humanized samples being very similar (0.4-0.5nM).

[0235] Example 8: Surface plasmon resonance (Biacore)-Affinity, association rate, and dissociation rate of an antibody with the mutation C102S in H330.

[0236] Evaluation of affinity constants by SPR

[0237] Immobilization Procedure

[0238] Running buffer (RB): HBS-EP+ consisting of 10 mM HEPES, 0.15 M NaCl, 3 mM EDTA, 0.05% v / v

[0239] Surfactant P20, pH 7.4, temperature: 25℃

[0240] Anti-human IgG (Fc) antibodies are chemically grafted onto the surface of a CM5 sensor chip using amine coupling according to the instructions in Cytivia 22064888AF. Briefly, the surface is first activated by injecting an NHS-EDC mixture. Then, several injections of anti-human IgG (25 μg / ml in immobilization buffer) are made with adjusted contact times (3 times over the 10 available on the kit). Finally, the surface is deactivated with ethanolamine 1M pH 8.5.

[0241] Immobilization of the investigated antibodies was performed sequentially at 5 μL / min on each flow cell until the immobilization level was between 90 and 100 RU. A 20 μg / mL MAb solution (MAb diluted in RB) was injected for 60 s, resulting in an immobilization signal between 850 and 2500 RU. When a high immobilization level was obtained, a regeneration solution (MgCl2 3M) was injected for 30 s, followed by a new injection with the adjusted concentration and contact time.

[0242] A negative control Ab is immobilized on the surface of sensor flow cell 1 (Fc1) and a positive control (chimeric 18B3 mAB) is immobilized on the surface of Fc2. 18B3 variant Abs are immobilized on the surfaces of Fc3 and Fc4:

[0243] [Table 8]

[0244] Single cycle kinetics (SCK) assay

[0245] Running buffer (HBS-EP+): 10 mM HEPES, 0.15 M NaCl, 3 mM EDTA, 0.05% v / v surfactant P20, pH 7.4, temperature: 25 °C. Flow rate: 30 μL / min.

[0246] Increasing concentrations (5; 10; 25; 50; 100 nM) of antigen (hβIG-H3) are injected for 180 seconds into each flow cell. A short dissociation in the RB is performed for each concentration of antigen. After injection of the highest concentration of antigen (100 nM), dissociation in the RB is recorded for 3600 seconds.

[0247] Three similar cycles with five RB injections were performed, followed by a cycle of antigen to perform the double subtraction procedure (blank run).

[0248] After three similar cycles with five RB injections and a dissociation time of 600 s, a blank run was performed to stabilize the system (start-up run).

[0249] Analysis in 1:1 interaction model:

[0250] [Table 9]

[0251] Conclusion:

[0252] The four mAbs examined have subnanomolar affinities (KD). The affinity of the humanized variant is only slightly less than that of the chimeric version. · There are no significant differences between the two variants (H-330 / L-228 and H-330 / L-41) either in the parental V1 version or in the mutated V1.2 version (C102→S102). For each variant, there is no significant difference between its parent version and its mutated version C102→S102. Very slight differences can be observed, which seem to be related to the experimental conditions, since the tests were performed in two series (i.e. with two SPR chips).

Claims

**Claim 1** A humanized anti-human βig-h3 monoclonal antibody or an antigen-binding fragment thereof, comprising a variable domain VH and a variable domain VL and specifically binding to an epitope of the βig-h3 protein (the epitope is shown as the sequence of SEQ ID NO: 16 or 30), wherein (a) the variable domain VH has a sequence shown as SEQ ID NO: 4 or 28; (b) it comprises a variable domain VL which is a humanized variant of the mouse 18B3 VL domain and has a sequence shown as SEQ ID NO: 18, and the humanized anti-human βig-h3 monoclonal antibody or an antigen-binding fragment thereof exhibits a thermal stability where DSC (Tm Fab) is 79 °C or higher, particularly between 79 or 80 and 83, 83.2, or 83.5 °C. **Claim 2** - a VH domain having a sequence shown as SEQ ID NO: 4 or 28; and - a VL domain having a sequence shown as SEQ ID NO: 10 or 13, the humanized anti-human βig-h3 monoclonal antibody or an antigen-binding fragment thereof according to claim 1. **Claim 3** A humanized anti-human βig-h3 monoclonal antibody or an antigen-binding fragment thereof, comprising a variable domain VH and a variable domain VL and specifically binding to an epitope of the βig-h3 protein (the epitope is shown as the sequence of SEQ ID NO: 16 or 30), wherein (a) - an H-CDR1 having a sequence shown as SEQ ID NO: 1; - an H-CDR2 having a sequence shown as SEQ ID NO: 2; - an H-CDR3 having a sequence shown as SEQ ID NO: 3 or 27 is included in the variable domain VH; (b) - an L-CDR1 having a sequence shown as SEQ ID NO: 7; - an L-CDR2 having a sequence shown as SEQ ID NO: 8; - an L-CDR3 having a sequence shown as SEQ ID NO: 9 is included in the variable domain VL, the humanized anti-human βig-h3 monoclonal antibody or an antigen-binding fragment thereof. **Claim 4** - a K that binds to the big-h3 protein at 5.8E-10 M or less, particularly between about 5E-10 and 5.8E-10 M, particularly about 5.35E-10 M Ｄ and / or - to the βig-h3 protein for about 6E-04 seconds －１ or more, particularly about 6.2E-04 to about 7E-04 seconds －１ between, particularly about 6.59E-04 seconds －１ of K ｄ to bind; and / or - having a stability where DSC (Tm Fab) is about 79 °C or higher, particularly between about 79 and about 83 °C, typically about 81.3 °C; and / or - having an excellent productivity of about 275 μg / ml in transient expression in CHO cells, the humanized anti-human βig-h3 monoclonal antibody or an antigen-binding fragment thereof according to any one of claims 1 to 3. **Claim 5** - a VH domain having a sequence shown as SEQ ID NO: 4 or 28; - A VH domain having the sequence shown as SEQ ID NO: 10 The humanized anti-human big-h3 monoclonal antibody or antigen-binding fragment thereof according to claim 3, comprising the same. **Claim 6** - A heavy chain comprising the variable domain and a CH constant domain, preferably a CH having the sequence shown as SEQ ID NO: 14; - A light chain comprising the variable domain and a CL constant domain, preferably a CL having the sequence shown as SEQ ID NO: 15 The humanized anti-human big-h3 monoclonal antibody or antigen-binding fragment thereof according to claim 3, comprising the same. **Claim 7** (a) - An H-CDR1 having the sequence shown as SEQ ID NO: 1; - An H-CDR2 having the sequence shown as SEQ ID NO: 2; - An H-CDR3 having the sequence shown as SEQ ID NO: 3 or 27 A variable domain VH comprising the same; (b) - An L-CDR1 having the sequence shown as SEQ ID NO: 11; - An L-CDR2 having the sequence shown as SEQ ID NO: 12; - An L-CDR3 having the sequence shown as SEQ ID NO: 9 The humanized anti-human big-h3 monoclonal antibody or antigen-binding fragment thereof according to claim 1, comprising a variable domain VL comprising the same. **Claim 8** - a K that binds to the βig-h3 protein at about 5E-10 M or less, particularly between about 4.5E-10 M and about 5E-10 M, typically about 4.76E-10 M Ｄ and / or - to the βig-h3 protein for about 5E-04 seconds －１ or more, particularly about 5.5E-04 to about 6E-04 seconds －１ between, particularly about 5.83E-04 seconds －１ of K ｄ to bind; and / or - Having a stability such that DSC (Tm Fab) is about 78 °C or higher, particularly about 78 - 82 °C, typically about 80.2 °C; and / or - Having a productivity of about 249 μg / ml in transient expression in CHO cells, the humanized anti-human big-h3 monoclonal antibody or antigen-binding fragment thereof according to claim 7. **Claim 9** - A VH domain having the sequence shown as SEQ ID NO: 4 or 28; The humanized anti-human big-h3 monoclonal antibody or antigen-binding fragment thereof according to claim 7 or 8, comprising a VL domain having the sequence shown as SEQ ID NO:

13. **Claim 10** - A heavy chain comprising the variable domain and a CH constant domain, such as a CH having the sequence shown as SEQ ID NO: 14; - A light chain comprising the variable domain and a CL constant domain, such as a CL having the sequence shown as SEQ ID NO: 15 The humanized anti-human big-h3 monoclonal antibody or antigen-binding fragment thereof according to claim 7 or 8, comprising the same. **Claim 11** A humanized anti-human big-h3 monoclonal antibody or an antigen-binding fragment thereof that comprises a variable domain VH and a variable domain VL and specifically binds to an epitope of the big-h3 protein (the epitope is shown as the sequence of SEQ ID NO: 16 or 30), - a VH domain having the sequence shown as SEQ ID NO: 6; - a humanized anti-human big-h3 monoclonal antibody or an antigen-binding fragment thereof that comprises a VL domain having the sequence shown as SEQ ID NO: 10 or 13.

12. Use of the humanized anti-human big-h3 monoclonal antibody or an antigen-binding fragment thereof according to any one of claims 1, 3, or 11 for the manufacture of a medicament for the treatment of cancer in a patient.

13. Use according to claim 12, wherein the cancer is a cancer in which the stromal protein big-h3 is expressed internally.

14. Use according to claim 12, wherein the cancer is pancreatic ductal adenocarcinoma (PDAC), lung cancer, head and neck cancer, colorectal cancer, bladder cancer, or melanoma.

15. A pharmaceutical composition comprising the humanized anti-human big-h3 monoclonal antibody or an antigen-binding fragment thereof according to any one of claims 1 or 11 and a pharmaceutically acceptable vehicle.

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