Bispecific antibodies that bind CD3 and ganglioside NGcGM3

Abstract

The present invention relates to the field of biotechnology and immunooncology. The invention discloses a bispecific antibody. The bispecific antibody comprises an antibody, an antibody fragment or a single-chain variable fragment for recognizing NGcGM3 ganglioside on tumor cells and an antibody, an antibody fragment or a single-chain variable fragment for recognizing a CD3 antigen on human immune effector cells. These bispecific antibodies are characterized in that they are capable of mediating selective cytotoxicity against NGcGM3-positive tumor cells, rather than normal cells capable of expressing the gangliosides, and allow recruitment of not only T lymphocytes but also NKT. The bispecific antibodies of the invention, as well as the nucleic acids encoding them, are useful for the treatment of lymphoproliferative diseases and solid tumors expressing NGcGM3.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and immuno-oncology. Specifically, it describes a bispecific antibody that recognizes the NGcGM3 antigen and CD3 molecule expressed in tumors and has the effector function of recruiting T cells, NKT cells, and Tγδ cells. Background Technology

[0002] T-cell conjugated bispecific antibodies (BAbs-T) have been increasingly recognized as one of the most promising alternatives for redirecting effector cells of the immune system to tumors, where they recognize antigens on the surface of malignant cells (Wu, Z. and N. Cheung (2018) Pharmacology therapeutics 182:161-175). Therefore, recruiting T lymphocytes to tumor cells, independent of the specific recognition of antigens by T-cell receptors, is a highly attractive and novel approach.

[0003] One of the challenges of this type of therapy is the appropriate selection of the tumor antigens it targets. Tumor-specific antigens, resulting from genetic and epigenetic changes, are the most selectively targeted antigens for tumors (Apavaloaei, A., et al. (2020) Cancers 12 (9):2607). Unfortunately, many of these antigens are intracellular antigens and are inaccessible to standard BAbs-T. Tumor-selective antigens include those overexpressed by tumors or differentially localized relative to normal tissues, and are also being utilized in BAbs-T-based therapies, but the potential for adverse reactions cannot be ruled out.

[0004] Specifically, BAbs-T for solid tumors has often shown safety concerns associated with cytokine release syndrome (CRS) and damage to normal tissues expressing the antigen. Preliminary results on BAbs-T for solid tumors report higher CRS rates (19-91%) than those used to treat hematologic malignancies (e.g., blinatumomab (7%-15%)) (Bendell, JC, et al. (2020); J Clin Oncol 38: 55-52; Borghaei, H., et al. (2020); Middleton, MR, et al. (2020); Clinical Cancer Research 26 (22): 5869-5878; Tran, B., et al. (2020). Annals of Oncology 31: S507). Off-target tumor toxicity has been a limiting factor in the use of BAbs-T against these types of tumors (Kobold, S., et al. (2018) Frontiers in Oncology 8:285).

[0005] Given the expression of NGcGM3 gangliosides in human tumors at different sites and their near absence in normal tissues, NGcGM3 gangliosides constitute a target for extensive research (Bardor, M., et al. (2005) Journal of Biological Chemistry 280(6): 4228-4237), and several therapies have been conceived based on this (Blanco, R., et al. (2011) International Scholarly Research Notices; Lahera, T., et al. (2014) Oncol). These include vaccines (e.g., Glycovax (Carr, A., et al. (2003). Journal of Clinical Oncology 21(6): 1015-1021), monoclonal antibodies (MAbs) (e.g., 14F7, characterized by its fine recognition of GM3 gangliosides, capable of distinguishing its N-glycosylated variant (which it recognizes) from its N-acetylated form (which it does not recognize) (Carr, A., et al. (2002). Hybridoma hybridomics 21(6): 463-468; Casadesús, AV, et al. (2013) Glycoconjugate 30: 687-699)), and their derived fragments (e.g., 3FM and 8Bhl (Rojas, G., et al. (2004). Journal of immunological methods 293(1-2): 71-83)). 14F7 antibodies were able to induce bloat-like cell death in mouse L1210 tumor cells (lymphoid-derived) but not in normal mouse lymphocytes (B and TCD4+). These differences in cytotoxicity can be attributed to the differential distribution of NGcGM3 gangliosides in plasma membrane lipid rafts between normal and tumor cells (Roque-Navarro, L., et al. (2008) Molecular Cancer Therapeutics 7(7): 2033-2041). However, these differences in ganglioside distribution between tumor and normal cells do not necessarily determine similar selectivity for other types of death mediated by the 14F7 binding domain and involving effector cells.

[0006] Given the non-protein nature of NGcGM3, therapeutic strategies such as MAbs and vaccines are inefficient in recruiting T cell responses and their cytotoxic capabilities against tumors expressing this ganglioside. This increases the need to develop therapies that mediate T lymphocyte cytotoxicity against NGcGM3-positive tumors. To date, there is no description of this ganglioside-specific BAb-T.

[0007] Since the first BAbs-T was developed, researchers have developed a variety of different antibody formats, ranging from low molecular weight designs to full IgG structures (Godar, M. et al. (2018). Expert Opinion on Therapeuticpatents 28(3):251-276).

[0008] While the chosen format can influence the function of these antibodies, it is generally accepted that the target molecule plays a central role in the potency and safety of BAbs-T (Li, H., et al. (2020) Cellular Molecular Immunology 17(5):451-461). This is influenced by the location of the identified epitope and the size of the target molecule. These characteristics play a fundamental role in the efficiency of immune synapse formation and subsequent T lymphocyte activation, and are determinants of the cytotoxicity of this type of antibody (Li, J., et al. (2017). Cancer Cell 31(3): 383-395). In summary, the nature of the target is crucial to ensuring the effectiveness of BAbs-T, as not every epitope can successfully recruit T lymphocytes through BAbs-T targeting it.

[0009] Although CD3-specific BAb-Ts can theoretically recruit not only T lymphocytes for cytotoxic activity but also NKT and Tγδ, there are few demonstrable examples of this ability. Specific BAb-Ts targeting human PDL1 have demonstrated their ability to induce cytotoxicity against PDL1+ tumor cells via peripheral blood NKT cells from healthy human donors (Horn, LA, et al. (2017) Oncotarget 8(35): 57964). To date, no BAb-T targeting any other molecular target has been described as possessing this property.

[0010] In view of the above-mentioned background technology, the inventors of this application have developed BAbs-T for cancer treatment, which recognizes NGcGM3 ganglioside, and this is the first description of BAbs-T with this antigen specificity.

[0011] The anti-NGcGM3 BAbs-T cells of this embodiment surprisingly achieved potent in vitro activity using human lymphocytes without prior activation, which particularly demonstrates their potent cytotoxic effects.

[0012] Furthermore, these BAbs-Ts are characterized by their ability to mediate selective cytotoxicity against NGcGM3-positive tumor cells, rather than normal cells, despite the fact that they may express this ganglioside. This provides a safety margin and minimizes off-target tumor effects. As a result, molecules with highly specific antitumor properties against malignant cells were obtained.

[0013] Furthermore, these anti-NGcGM3 BAbs-Ts allow recruitment not only of T lymphocytes but also of NKT cells and induce tumor cell lysis, and are the first BAbs-Ts to exhibit this specificity. Summary of the Invention

[0014] In one embodiment, the present invention relates to BAbs-T, which comprises an antibody, antibody fragment, or single-chain variable fragment (scFv) recognizing NGcGM3 gangliosides on tumor cells, and an antibody, antibody fragment, or scFv recognizing human CD3 molecules. Specifically, the antibody recognizing NGcGM3 is human IgG, the sequence of which is selected from the group consisting of SEQ ID NOs 1-4 or variants of these sequences having more than 90% identity with them.

[0015] These BAbs-Ts can have a format selected from the group containing the following: TandAb, DART, DART-Fc, DuoBody, CrossMab, KiH, BiTE, Triomab, IgG-scFv.

[0016] In one specific implementation, the heavy chain of an antibody that recognizes NGcGM3 is fused directly or via a linker to an antibody, antibody fragment, or scFv targeting human CD3.

[0017] In another embodiment, the antibody, antibody fragment, or scFv that recognizes the human CD3 molecule has a sequence selected from the group consisting of SEQ ID NO. 5-7, or variants of these sequences that have more than 90% identity with said sequence.

[0018] In another specific embodiment, the antibody, antibody fragment, or scFv that identifies NGcGM3 has a heavy chain variable region selected from the group consisting of SEQ ID NO. 10 and SEQ ID NO. 15, or variants thereof having more than 90% identity with these sequences; and the antibody, antibody fragment, or scFv that identifies NGcGM3 has a light chain variable region selected from the group consisting of SEQ ID NO. 11-14, or variants thereof having more than 90% identity with these sequences.

[0019] In another embodiment, the present invention relates to a pharmaceutical composition comprising BAbs-T as described herein as an active ingredient and a pharmaceutically acceptable carrier.

[0020] In yet another embodiment, the present invention relates to the use of BAbs-T in the treatment of lymphoproliferative disorders and solid tumors expressing NGcGM3. In particular, it relates to the use of nucleic acids encoding BAbs-T in gene therapy for tumors expressing NGcGM3, said gene therapy being based on the injection of mRNA or transduction particles encoding BAbs-T.

[0021] In another embodiment, the present invention relates to a method of treating a subject in need, comprising administering the disclosed BAbs-T in a dose range of 20ug-10mg via subcutaneous, intravenous, intradermal, intramuscular, intratumoral, or intraperitoneal routes. Invention Details

[0023] Description of BAbs-T

[0024] This invention relates to bispecific antibodies or multifunctional fusion proteins comprising antibodies, antibody fragments or scFv that recognize NGcGM3 gangliosides on tumor cells, and antibodies, antibody fragments or scFv that recognize human CD3 molecules.

[0025] The term T-cell binding bispecific antibody (BAb-T) used in this invention refers to a class of bispecific antibodies that can bind to the cytotoxic effects of T cells and NKT cells and recruit tumor cells expressing specific antigens.

[0026] The terms fusion protein and BAb-T are used interchangeably in this invention.

[0027] These BAbs-Ts can have different formats well known to those skilled in the art, and can be obtained using the variable domains and / or CDR sets described herein, and through conventional techniques (Godar, M. et al. (2018). Expert Opinion on Therapeutic patents 28(3):251-276). The format of the fusion protein of this invention ensures the recruitment, activation, and cytotoxicity of T lymphocytes and NKT cells to tumor cells.

[0028] According to one embodiment of the present invention, the BAbs-T described herein adopts a structure based on intact IgG.

[0029] In one specific implementation, the IgG backbone is based on human IgG1 and contains a set of modifications that eliminate or reduce FcγR binding and complement fixation, and reduce its potential to recruit effector functions associated with its Fc region. This modified Fc prevents cross-linking of the Fcγ-CD3 receptor (caused by antibody binding to sites different from tumor targets), thereby preventing undesirable effects on other cells of the immune system mediated by T cells, which limits the efficacy of BAbs-T (Labrijn, AF, et al. (2019) Nature reviews Drug discovery 18(8): 585-608).

[0030] In particular, these BAbs-T human IgG1s have modifications in the CH2 domain (SEQ ID NO. 1) that eliminate or reduce FcγR binding and complement fixation (Wines, BD, et al. (2000) The Journal of Immunology 164(10): 5313-5318; Armour, KL, et al. (2003) Molecular immunology 40(9): 585-593; Sazinsky, SL, et al. (2008) Proceedings of the National Academy of Sciences 105(51): 20167-20172; Schlothauer, Herter et al. (2016) Protein Engineering, Design 29(10): 457-466).

[0031] Optionally, the modified human IgG1 backbone has the N297X mutation (SEQ ID NO. 2), where X is selected from the group consisting of alanine, glycine, and glutamine. This mutation eliminates the canonical glycosylation site in the Fc region of human IgG1 (Chao, DT, et al. (2009) Immunological investigations 38(1): 76-92), thereby deriving a non-glycosylated immunoglobulin in this region that does not have the ability to bind to the Fcγ receptor (Wang, L.-X., et al. (2019) Annual review of biochemistry 88: 433-459). Furthermore, the IgG backbone contains variants that have more than 90% identity with SEQ ID NO. 1 and 2.

[0032] In one specific implementation, the human IgG backbone is IgG2 (SEQ ID NO. 3, Uniprot P01859) or IgG4 (SEQ ID NO. 4, Uniprot P01861), which do not have the ability to bind to the Fcγ receptor (Vafa, O., et al. (2014) Methods 65(1): 114-126; Gillies, SD, et al. (1999) Cancerresearch 59(9):2159-2166; Newman, R., et al. (2001) Clinical Immunology 98(2):164-174), as well as variants having more than 90% identity with the said sequence.

[0033] Optionally, the design of the BAbs-T target of the present invention is based on a tetravalent molecule of intact IgG with minimized or no effector function, having two NGcGM3 ganglioside binding domains and two CD3 binding domains.

[0034] In another embodiment, the BAbs-T anti-NGcGM3 obtained in this invention is characterized by having an anti-CD3 scFv, which is directly or via a linker of 10-27 amino acid residues linked to the carboxyl terminus of the anti-NGcGM3 antibody heavy chain (IgG-scFv format). In particular, the scFv of the anti-CD3 antibody is bound to humanized UCHT1 (Zekri, L., et al. (2021) EMBO molecular medicine 13(2):e11902, SEQ ID NO.5), humanized OKT3 (Adair, JR, et al (1994) Human Antibodies 5(1-2):41-47; SEQ ID NO.6) or L2K (US9611325B2, SEQ NO. ID 7), or variants of these sequences having more than 90% identity with these sequences.

[0035] Furthermore, the BAbs-T of the present invention can adopt the format described in the prior art, such as BiTE (WO2005061547, Frankel and Baeuerle, 2013), Triomab (WO1995033844, Lindhofer, H., et al. (1995) Journal of immunology 155(1):219-225; Tandem diAbody (TandAb) (Reusch, U., et al. (2015) MAbs 7(3): 584-604; Dual AffinityRetargeting (DART) / Dual affinity Retargeting-Fc (DART-Fc) (Chichili GR et al. (2015) Sci Transl Med 7(289): 289ra82), DuoBody (Gaudet, F., et al. (2016) Blood 128(22):2824) and CrossMab and / or mortar and pestle structure (KiH) (WO201326833; Bacac, M., et al. (2016) Clinical Cancer Research 22(13):3286-3297).

[0036] The BAbs-T described in this invention has an affinity range (KD in the range of 10). -7 Up to 10 -10 (between M and M) recognizes NGcGM3 gangliosides and uses KD at 10-7 Up to 10 -10 The M-range has an affinity for binding human CD3 molecules, which allows for the activation of T cells or other cells expressing CD3 clusters.

[0037] The BAbs-T antibodies described in this article recruit T cells through their CD3-specific scFv domain. Furthermore, these antibodies exhibit the property of recruiting and activating NKT cells, thereby mediating the lysis of tumor cells expressing NGcGM3.

[0038] In one specific embodiment, the light chain constant region of BAbs-T is characterized by belonging to κ (Uniprot P01834) or human λ isotype (Uniprot P0CG04) and having sequences corresponding to SEQ ID NO. 8 and SEQ ID NO. 9, respectively, as well as variants having more than 90% identity with said sequences.

[0039] On the other hand, the variable region of the antibody heavy chain can be murine, humanized, or fully human, and originates from an immunoglobulin capable of recognizing NGcGM3 gangliosides. The variable region is identified as, but not limited to, SEQ ID NO 10, including variants containing any of the following mutations:

[0040] Position 5: Q replaces V

[0041] Position 9: N replaces A

[0042] Position 11: L replaces V

[0043] Position 12: A replaces V

[0044] Position 18: M replaces V

[0045] Position 19: K replaces R

[0046] Position 120: M replaces V

[0047] Position 40: R replaces A

[0048] Position 42: D replaces G

[0049] Position 48: I replaces V

[0050] Furthermore, the variable region of the antibody light chain can be murine, humanized, or fully human. Such variable regions are identified as substituting, but not limited to, SEQ ID NO 11, including variants containing any of the following mutations:

[0051] Light chain

[0052] Position 39: R replaces K

[0053] Position 40: T replaces P

[0054] Position 41: H replaces G

[0055] Position 42: E replaces Q

[0056] Position 58: I replaces V

[0057] In addition, these light chain variable regions include SEQ ID NO 12 and SEQ ID NO 13.

[0058] Pharmaceutical Composition

[0059] The BAbs-T targets of the present invention can exist as active ingredients, forming part of various pharmaceutical compositions suitable for them, and pharmaceutically acceptable carriers. The concentration of the active ingredient in the pharmaceutical composition is in the range of 0.5 mg / ml to 20 mg / ml, preferably 1 mg / ml to 10 mg / ml, or lyophilized.

[0060] Pharmaceutically acceptable carriers include, but are not limited to, saline, pH-neutral phosphate-buffered saline, and other similar substances. Other buffers, dispersants, and non-toxic, inert substances suitable for patient delivery may be included in the compositions of the present invention. The compositions may be solutions suitable for administration and are generally sterile and free of unwanted particles.

[0061] Therapeutic applications and treatment methods

[0062] This invention relates to the use of BAbs-T disclosed herein in the treatment of NGcGM3-positive tumors. Specifically, it relates to the use in the treatment of lymphoproliferative disorders such as chronic lymphocytic leukemia (CLL) and multiple myeloma, as well as advanced solid tumors expressing NGcGM3 such as head and neck tumors, brain tumors, adult and pediatric gliomas, pancreatic cancer, esophageal and breast tumors, non-small cell lung cancer, and tumors of the nasopharynx, stomach, bladder, colorectal region, and melanoma. Additionally, it relates to the use of gene therapy methods for treating the aforementioned malignancies by subcutaneous, intramuscular, or intratumoral injection of mRNA or transduction particles encoding BAbs-T of the present invention.

[0063] In another embodiment, the present invention relates to a method of treating a subject in need, comprising administering a pharmaceutical composition comprising any of the BAbs-T described herein as an active ingredient via subcutaneous, intravenous, intradermal, intramuscular, intratumoral, or intraperitoneal route, at a dose ranging from 20 μg to 10 mg. Specifically, the administration of the pharmaceutical composition is performed for 1-13 cycles, with 1-7 infusions per cycle, over a 21- or 28-day cycle.

[0064] These molecules, in addition to mediating cytotoxic effects on tumor cells, also possess immunomodulatory properties, thus supporting their potential combination with other immunotherapies for patients with various types of cancer. Similarly, they may also be used in combination with other immunomodulators and classic cancer therapies such as chemotherapy and radiotherapy. In this way, the mentioned BAbs-Ts aim to constitute a therapeutic direction for cancer treatment. This effect will be associated with the selective aggregation of immune effector cells (such as T and NKT lymphocytes) against the cytotoxic effects of NGcGM3-expressing tumor cells, leading to delayed tumor growth and higher survival rates in treated individuals. Furthermore, there is the ability to induce a controlled inflammatory cytokine environment, which helps activate other immune effectors, thereby enhancing anti-tumor effects. Simultaneously, it is important to emphasize the low toxicity levels they exhibit in animal models against healthy tissues expressing NGcGM3 gangliosides, supporting their good safety profile in patient use.

[0065] All of this translates into greater hope and a higher quality of life for the patients being treated. Attached Figure Description

[0066] Figure 1 : Represented in the format of anti-NGcGM3 BAbs-T.

[0067] Figure 2 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of antibodies 14F7hT-UCHT1 (A, lanes 1 and 2), 3Fm-UCHT1 (A, lanes 3 and 4), 14F7hT (A, lanes 5 and 6; B and C, lanes 1), and 14F7hT-2c11 (B and C, lanes 2). Non-reducing conditions (A: lanes 1, 3, 5; B: lanes 1 and 2). Reducing conditions (A: lanes 2, 4, 6; C: lanes 1 and 2).

[0068] Figure 3 : NGcGM3 on the surface of P3X63 and L1210 tumor cell lines was recognized by BAbs-T (14F7hT-UCHT1 and 3Fm-UCHT1) (A) and 14F7hT-2c11 (B), and CD3 on the surface of Jurkat cell line was recognized by BAbs-T (14F7hT-UCHT1 and 3Fm-UCHT1) (C).

[0069] Figure 4 Cytotoxic activity against P3X63 tumor cell lines mediated by BAbs-T 14F7hT-UCHT1 (A and B), 3Fm-UCHT1 (A) and 14F7hT-2c11 (C) in the presence of effector cells.

[0070] Figure 5 Cytotoxic activity of mouse NKT cells against the P3X63 tumor cell line mediated by the 14F7hT-2c11 antibody. Purified NKT cells (A) and NKT hybridomas (B) were used as effector cells.

[0071] Figure 6 The antitumor effect of 14F7hT-2c11 in the BALB / c-X63 treatment model was measured on day 7 after tumor growth.

[0072] Figure 7 T cells were activated by the 14F7hT-UCHT1 antibody in the presence of P3X63 tumor cell lines CD8+(A) and CD4+(B).

[0073] Figure 8 Induction of T lymphocyte proliferation mediated by BAbs-T 14F7hT-UCHT1 (A and B) and 14F7hT-2c11 (C) in the presence of P3X63 tumor cell line.

[0074] Figure 9 : Specificity of cytotoxic effects on tumor cells mediated by human effector cells and 14F7hT-UCHT1.

[0075] Figure 10 The absence of cytotoxicity of BAbs-T 14F7hT-2c11 (A) and 14F7hT-UCHT1 (B) on normal cells (mouse lymphocytes).

[0076] Figure 11 The evaluation of in vivo T cell activation mediated by BAbs-T 14F7hT-UCHT1 (A) and 14F7hT-2c11 (B) in the presence of normal tissues expressing this ganglioside was based on the serum IFNγ levels in treated animals.

[0077] Figure 12 Evaluation of in vivo toxicity of BAbs-T 14F7hT-UCHT1 based on body weight (A) and relative organ weight (B).

[0078] Figure 13 Evaluation of the in vivo toxicity of BAbs-T 14F7hT-2c11 based on weight measurements.

[0079] The invention is further illustrated by the following embodiments and accompanying drawings. However, these embodiments should not be construed as limiting the scope of the invention.

[0080] The specific method

[0081] Example 1. Design and obtaining a genetic construct encoding BAbs-T.

[0082] Human BAbs-T, composed of multifunctional molecules, was designed based on the format of intact IgG (IgG-scFv) with bivalent binding to NGcGM3 and human CD3. Specifically, BAbs-T (14F7-UCHT1, 3FM-UCHT1, and 8Bhl-UCHT1) correspond to... Figure 1 The format shown.

[0083] To mimic the activity of human BAbs-T in immune-active mice, BAbs-T 14F7-2c11 was designed, exhibiting the same specificity for tumor antigens and the same format as human BAbs-T. Its format is as follows: Figure 1 As shown, it exhibits bivalent binding to NGcGM3 and mouse CD3.

[0084] The antigen-binding domains of BAbs-T (14F7hT-UCHT1 and 14F7hT-2c11) contain the variable regions (VL and VH) of 14F7hT Mab (Fernández-Marrero, Y., et al. (2011) Immunobiology 216(12):1239-1247, SEQ ID NO. 14 and 15, respectively); the antigen-binding domain of 3Fm-UCHT1 retains the VH region of 14F7hT (SEQ ID NO 15) and shares the VL region of the scFv3Fm fragment (SEQ ID NO. 12), while the antigen-binding domain of 8Bhl-UCHT1 retains the VH region of 14F7hT MAb (SEQ ID NO. 15) and shares the VL region of the scFv8Bhl fragment (SEQ ID NO. 13). BAbs-T (14F7hT-UCHT1, 3Fm-UCHT1, 8Bhl-UCHT1) fused a humanized UCHT1 MAb specific to human CD3 to the C-terminus of the heavy chain (Zekri, L., et al. (2021) EMBO molecularmedicine 13(2):e11902, SEQ. ID 5), while 14F7hT-2c11 fused a mouse CD3-specific scFv 2c11 to the same site (SEQ. ID 16, Fernandes, RA, et al. (2012). Journal of Biological Chemistry 287 (16): 13324-13335). Furthermore, the human IgG1 backbone used in the four BAbs-Ts was identified using SEQ. ID NO. 1.

[0085] On the other hand, the light chains of anti-NGcGM3 BAbs-T (14F7-UCHT1, 3FM-UCHT1, 8Bhl-UCHT1 and 14F7hT-2c11) have human Cκ constant regions (SEQ. ID NO. 8).

[0086] To obtain genetic constructs encoding the BAbs-T light chain, the VL3Fm and VL14F7hT genes were cloned into the pGH1.2_VκPSMA_Cκh vector. Thus, the genetic constructs pGH1.2_VL3Fm_Cκh and pGH1.2_VL14F7hT_Cκh were obtained. The first was used to obtain 3FM-UCHT1, while the second was used to generate BAbs-T 14F7-UCHT1 and 14F7hT-2c11. To obtain genetic constructs encoding the heavy chains of BAbs-T 14F7-UCHT1 and 3FM-UCHT1, the VH gene of AcM 14F7hT was cloned into the vector pGH1.2-IgGscFv_PSMA_UCHT1 (modified IgG1h-scFvUCHT1).

[0087] To obtain the genetic construct encoding the heavy chain 14F7hT-2c11, the scFv 2c11 gene from the construct pGH1.2-IgGscFv_10B3_2c11 was cloned into the previously obtained vector pGH1.2-IgGscFv_14F7hT_UCHT1.

[0088] Example 2. Anti-NGcGM3 BAbs-T as a complete and functional protein expression.

[0089] Transient expression of BAbs-T was performed in suspension ExpiCHO-S cells using the genetic construct described above. Ten days after transfection, the recombinant molecules present in the supernatant were purified using a sequential protocol consisting of a first step of protein A affinity chromatography followed by size exclusion chromatography.

[0090] Next, the size and integrity of BAbs-T were confirmed by SDS-PAGE and subsequent Coomassie Brilliant Blue staining. For BAbs-T 14F7hT-UCHT1 and 3Fm-UCHT1, SDS-PAGE was performed on a 10% polyacrylamide gel using both reducing and non-reducing conditions. Figure 2 A). For the 14F7hT-2c11 antibody, at 7.5% ( Figure 2 B) and 12% Figure 2 SDS-PAGE was performed on a polyacrylamide gel (C) under both non-reducing and reducing conditions. 14F7hT MAb was used as a monospecific antibody control. Figure 2As observed, under non-reducing conditions, purified BAbs-T produced a band migrating to the expected size, equivalent to approximately 204 kDa. 14F7-hT MAb, as expected, exhibited greater electrophoretic mobility due to its molecular weight of approximately 150 kDa. The experimental reducing conditions revealed the presence of both chains of the recombinant bispecific molecule, with migration corresponding to theoretical sizes: 77 kDa for the heavy chain and 25 kDa for the light chain. Figure 2 ).

[0091] The control monospecific antibody 14F7hT MAb also showed the presence of bands corresponding to these two chains, at the expected size: a 50 kDa band for the heavy chain and a 25 kDa band for the light chain. Figure 2 ).

[0092] Example 3. BAbs-T recognizes NGcGM3 and CD3 molecules.

[0093] To determine the dual recognition of the generated antibody, its reactivity to gangliosides NGcGM3 and CD3 molecules was evaluated.

[0094] The recognition of gangliosides was evaluated by binding antibodies 14F7hT-UCHT1 and 3Fm-UCHT1 to mouse tumor cell lines L1210 and P3X63, which express high levels of NGcGM3 in their plasma membranes (Carr, A., et al. (2002) Hybridoma hybridomics 21(6):463-468; Roque-Navarro, L., et al. (2008) Molecular Cancer Therapeutics 7(7):2033-2041). This reactivity was determined by flow cytometry, with antibodies used at equimolar concentrations. Figure 3 As seen in Figure A, BAbs-T 14F7-UCHT1 and 3Fm-UCHT1 demonstrated their ability to recognize these two tumor cell lines in a concentration-dependent manner, as did the 14F7hT antibody used as a positive control. This confirms that the antigen-binding properties of the generated recombinant molecules in the plasma membrane context are not impaired by the presence of scFv coupled to the C-terminus of the heavy chain when compared with classical IgG of equivalent specificity. As expected, no recognition of these cells by CC-1 (used as an allotype control, BAb-T) was observed.

[0095] In the case of 14F7hT-2c11 antibody ( Figure 3B) The binding of the antibody to mouse tumor cell lines P3X63 and L1210 was also evaluated. This recognition was confirmed by flow cytometry using the antibody at 5 nM. Its ability to bind to these two tumor cell lines was demonstrated, as was the case with the 14F7hT antibody used as a positive control. As expected, no binding was observed with the BAb-T MOPC-UCHT1 antibody used as an isotype control.

[0096] Given that 14F7hT-UCHT1 and 3Fm-UCHT1 are specific for ganglioside NGcGM3 and human CD3 molecules, it is also important to determine their ability to interact with the latter as part of a reactivity study. For this purpose, their recognition of the human T lymphocyte cell line Jurkat (CD3-positive) was evaluated by flow cytometry. Antibodies were used in equimolar amounts at different concentrations. Figure 3 C showed that BAbs-T 14F7hT-UCHT1 and 3Fm-UCHT1 bound to this cell line in a dose-dependent manner, similar to CC-1, which has the same format and the same amino acid sequence relative to the CD3 binding site (positive control).

[0097] Example 4. Anti-NGcGM3 BAbs-T induces cytotoxicity in ganglioside-positive tumor cell lines.

[0098] Given previous results demonstrating the recognition properties of the 3Fm-UCHT1 and 14F7hT-UCHT1 antibodies, it was decided to evaluate their ability to mediate cytotoxicity in NGcGM3-positive tumor cell lines. Specifically, for this measurement, P3X63 cells were selected as target cells (T), and inactivated peripheral blood mononuclear cells (PBMCs) purified from healthy human donors were selected as effector cells (E), co-cultured at a ratio of 5:1 (E:T). The 3Fm-UCHT1 and 14F7hT-UCHT1 antibodies were used at concentrations ranging from 0.15 to 50 nM, and CC-1 was used as an irrelevant antibody (negative control) at 50 nM, given the binding sites with the target cells (recognizes the prostate-specific membrane antigen PSMA and the human CD3 molecule, Zekri, L., et al. (2021) EMBO molecular medicine 13(2):e11902). Dead cells were stained with 7-AAD reagent. Cell lysis was measured by flow cytometry after 72 hours. For each experimental condition, count the number of surviving target cells (7-AAD- / CD45-) treated with BAbs-T, and calculate the percentage of specific lysis using the following formula: Specific lysis (%) = 100 - ((number of surviving target cells (+BAb-T)) / (number of surviving target cells (-BAb-T)) × 100). Perform two replicate evaluations for each experimental condition.

[0099] exist Figure 4 In A, high cytotoxicity (greater than 95%) induced by the 14F7hT-UCHT1 and 3Fm-UCHT1 antibodies was observed even at low concentrations, such as 0.15 nM, in the presence of effector cells. The specificity of this effect was supported by the marginal cytotoxicity (20%) of the irrelevant antibody CC-1 (negative control), evaluated at the highest concentration tested against anti-NGcGM3 BAbs-T. As a qualitative measure of this effect, such as... Figure 4 As shown in B, P3X63 (target) tumor cells, characterized as larger size, are visualized under an optical microscope. Compared to treatment with an irrelevant bispecific antibody (right panel), as described above, treatment with 14F7hT-UCHT1 (15 nM) in the presence of human effector cells significantly reduced the number of target cells (left panel).

[0100] The ability of 14F7hT-2c11 to induce cytotoxicity was evaluated on P3X63 mouse myeloma cells (target cells), with purified T lymphocytes pre-labeled with CFSE from the spleen and lymph nodes of C57BL / 6 mice used as effector cells. Antibodies were used at 15 nM, 5 nM, and 0.5 nM in the cytotoxicity assay. 14F7hT-UCHT1 (which recognizes human CD3) was used as an irrelevant antibody (negative control) based on its binding site with effector cells. Dead cells were stained with LIVE / DEADFixableNear-IR Deadcell reagent. Cell lysis was measured by flow cytometry after 72 hours, and cell lysis was calculated using the formula described above based on the number of surviving target cells (LIVE / DEAD FixableNear-IR Deadcell- / CFSE-) treated with or without BAbs-T.

[0101] like Figure 4 As shown in C, the bispecific antibody 14F7hT-2c11 was able to lyse up to approximately 96% of target cells in the presence of mouse effector cells. Furthermore, given that an unrelated antibody (14F7hT-UCHT1) binding to mouse T lymphocytes did not produce this biological effect, this supports the specificity of the cytotoxicity induced by mouse BAb-T.

[0102] Example 5. BAb-T14F7hT-2c11 induces NKT cell-mediated cytotoxicity in NGcGM3 positive cell lines.

[0103] Although theoretically, the binding of BAbs-T to CD3 molecules could also recruit NKT and Tγδ, this has only been documented for BiTE-format antibodies, which are well-known for their high potency (Horn, LA, et al. (2017) Oncotarget8(35):57964). For this reason, it was decided to evaluate whether antibodies in the complete tetravalent IgG format (e.g., the antibody carried by the anti-NGcGM3 BAbs-T of this invention) could recruit NKT lymphocytes for effector activity against NGcGM3-positive cells.

[0104] For this purpose, in the first method, the ability of 14F7hT-2c11 to mediate iNKT cell-induced lysis of NGcGM3-positive tumor cells was investigated. In this experiment, P3X63 (0.5 x 10⁻⁶) cells were used. 5 Incubation was performed using iNKT cells purified from spleen cells of C57BL / 6 mice as target cells (T) and as effector cells (E). Figure 5A) or FF13 iNKT cell hybridoma (Schümann, J., et al. (2007). European journal of immunology 37 (6): 1431-1441)( Figure 5 B), pre-labeled with CFSE, was co-cultured at a 10:1 ratio (E:T). BAbs-T was used at 15 nM. Cell lysis was measured by flow cytometry after 72 hours. For each experimental condition, the percentage of surviving target cells (LIVE / DEAD Fixable Near-IR Deadcell- / CFSE-) was calculated relative to the untreated tumor cell condition using the formula described above. Each experimental condition was evaluated twice in duplicate. 14F7hT-UCHT1, which does not recognize mouse CD3 molecules, was used as a negative control.

[0105] exist Figure 5 In both A and B, the 14F7hT-2c11 antibody was observed to induce cytotoxicity in P3X63 target cells in the presence of iNKT cells, resulting in a reduction of more than 50% of viable target cells. This result supports another mechanism of action of BAbs-T, which has been rarely addressed in existing technologies and has not been previously described for this target.

[0106] Example 6. 14F7hT-2c11 has an anti-tumor effect on NGcGM3-positive tumors implanted in immune-active mice.

[0107] The clinical development of BAbs-T has been accompanied by the creation of similar molecules, but with a specific targeting of tumor antigens and mouse CD3, in order to mimic the effects of such biopharmaceuticals in immune-active animals (Labrijn, AF, et al. (2019) Nature reviews Drug discovery 18(8): 585-608; Benonisson, H., et al. (2019) Molecular cancer therapeutics 18(2): 312-322).

[0108] Therefore, the antitumor effect of the bispecific antibody 14F7hT-2c11 was measured in BALB / c (immunely active) mice in a P3X63 treatment model to demonstrate its ability to eliminate malignant cells in vivo and in vitro, as previously tested. For this purpose, 1×10⁻⁶ cells were used. 6 One P3X63 cell was seeded into the right groin of the animal. Once the tumor was palpable (not measurable: 65 mm), the tumor was detected. 3 or 78 mm 3Treatment began on day 1. Treatment was administered intraperitoneally on days 1, 4, and 7 with 12.5 μg of 14F7-2c11 in 200 μL of PBS.

[0109] like Figure 6 As shown, 7 days after tumor implantation, the group treated with 14F7hT-2c11 showed smaller tumor volume compared with the control group (treated with PBS) (Tukey test, p<0.05).

[0110] This experiment provides evidence for the in vivo antitumor activity of 14F7hT-2c11, which validates the therapeutic potential of BAbs-T with the variable region of 14F7hT and the above-described format in the context of immune-active individuals.

[0111] Example 7. Anti-NGcGM3 BAbs-T promotes T cell activation.

[0112] In addition, as part of the bioactivity of anti-NGcGM3 BAbs-T, it was decided to assess the activation status of T lymphocytes. To this end, PBMCs (the source of effector cells) from healthy human donors (P3X63 cells) were co-incubated at a 5:1 ratio for 72 hours in the presence of different concentrations of bispecific antibody, and the activation marker CD69 was measured by flow cytometry. As a negative control, the same assay was performed by co-incubating PBMCs with anti-ganglioside BAb-T in the absence of tumor cells.

[0113] Based on the expression of the early activation antigen CD69, 14F7hT-UCHT1 showed inhibition of CD4+ ( Figure 7 A) and CD8+ T cells ( Figure 7 B) Concentration-dependent effect of activation. In contrast, cells treated with BAb-T CC-1 (negative control) showed baseline activation percentage values ​​similar to those of untreated cells, which also supports the specificity of this effect.

[0114] In the presence of target cells, BAbs-T stimulated the activation and proliferation of T lymphocytes, thereby promoting better anti-tumor effects. Therefore, we evaluated whether 14F7hT-UCHT1 could promote the proliferation of CD4+ and CD8+ T cell populations by analyzing PBMCs (2.5 x 10⁻⁶ cells) labeled with CTV reagent. 5The cells were co-incubated with the NGcGM3-positive tumor cell line P3X63 at a 5:1 (E:T) ratio and with different concentrations of 14F7hT-UCHT1 antibody. Bispecific CC-1 (15 nM) was used as a negative control, while PHA (10 μg / mL) was used as a proliferation positive control. After 72 hours, the CTV signal dilution of each T cell subset, indicating their proliferation, was measured by flow cytometry.

[0115] 14F7hT-UCHT1 induced the proliferation of human T lymphocytes, preferentially CD8+ T subsets. Figure 8 A), this depends on the concentration of BAb-T. At a maximum concentration of 15 nM for 14F7hT-UCHT1, 25% of the initially dye-labeled total CD8+ T lymphocytes were able to proliferate. Similar but milder behavior was observed in the CD4+ T lymphocyte population. Figure 8 B), at the same concentration, 6% proliferation was verified. As expected, maximum proliferation was obtained in cells treated with the mitogen PHA (used as a positive control). On the other hand, the unrelated BAb-T CC-1 (negative control) did not produce this biological effect on T lymphocytes, and the percentage values ​​obtained were similar to those in untreated cells.

[0116] Similarly, we set out to evaluate whether 14F7hT-2c11 (an alternative to 14F7hT-UCHT1) could promote the proliferation of mouse T cell populations. For this purpose, total lymphocytes previously labeled with CFSE from C57BL / 6 mouse lymph nodes were co-incubated with the NGcGM3-positive tumor cell line P3X63 at a ratio of 10:1 (E:T) and 15 nM of the 14F7hT-2c11 antibody. Bispecific 14F7hT-UCHT1 (15 nM) was used as a negative control because it does not recognize mouse T lymphocytes. After 72 hours, the CFSE signaling dilution of T cells, indicating their proliferation, was measured by flow cytometry. Cell viability was measured using LIVE / DEADFixableNear-IR Deadcell. Starting with LIVE / DEAD FixableNear-IR Deadcell- / CFSE+ cells, T cell proliferation was assessed by quantifying the percentage of T lymphocytes with altered mean fluorescence intensity (MFI) labeled with CFSE reagent in the presence of P3X63 cells under different treatments. Each experimental condition was evaluated in triplicate.

[0117] like Figure 8As shown in C, using 15 nM of the 14F7hT-2c11 antibody, 45% of the total T lymphocytes initially labeled with CFSE dye were able to proliferate. In contrast, the irrelevant antibody 14F7hT-UCHT1 did not produce this biological effect on T lymphocytes, and the percentage values ​​obtained were similar to those of untreated cells (Student's t-test, p < 0.05).

[0118] This set of experiments shows that anti-NGcGM3 BAbs-T can induce T lymphocyte proliferation, which helps to amplify the biological effects of these antibodies.

[0119] Example 8. Anti-NGcGM3 BAbs-T only induces cytotoxicity against tumor cells and has no effect on normal NGcGM3-positive cells.

[0120] To confirm the specificity of anti-NGcGM3 BAbs-T cells in exerting tumor cell-mediated cytotoxic effects in the presence of effector cells, a study was conducted using the L1210 tumor cell line and its L1210 cmah-kd variant as target cells (T). In this experiment, we performed the study in a similar manner to the one described above, using a 2:1 (E:T) ratio and PBMCs from healthy human donors as the effector cell source. Figure 9 As shown, 14F7hT-UCHT1 induced dose-dependent cytotoxicity in the L1210 cell line, while the viability of L1210 cmah-kd cells was almost unaffected by this treatment. The percentage of cytotoxicity in these cells was similar to that in L1210 cells treated with the CC-1 antibody (negative control). This result confirms the specificity of the antibody-induced cytotoxic effect.

[0121] Furthermore, the high potency of BAb-T (14F7hT-UCHT1) was demonstrated here, achieving over 50% cytotoxicity using only 10 pM of the antibody and unactivated effector cells.

[0122] Although the presence of gangliosides is essential for the cytotoxic effects of anti-NGcGM3-mediated BAb-T cells on T lymphocytes, the difference between normal and tumor cells for this effect is not significant. As evidence of the preferential cytotoxic effects of anti-NGcGM3-mediated BAb-T cells on tumor cells compared to normal cells that express this ganglioside, its lytic effect on normal mouse lymphocytes was evaluated, in which a subset, predominantly CD4+ cells, is known to express this ganglioside and be recognized by the 14F7 antibody (Roque-Navarro, L., et al. (2008) Molecular Cancer Therapeutics 7(7): 2033-2041).

[0123] To examine whether BAb-T 14F7hT-2c11 could induce cytotoxicity in these normal cells in vitro, total lymphocytes (0.5 x 10⁻⁶) from the lymph nodes of C57BL / 6 mice were used. 6 The cells were incubated with the bispecific antibody at different concentrations for 72 hours. In this case, T lymphocytes acted as effector cells and were also potential target cells due to the presence of gangliosides. For each experimental condition, the percentage of live target cells (LIVE / DEAD Fixable Near-IR Deadcell) was calculated by flow cytometry. Figure 10 As shown in Figure A, mouse lymphocytes treated with different concentrations of 14F7-2c11 antibody showed no difference in viable cell percentage compared to cells incubated with 14F7-UCHT1 (negative control, which does not recognize mouse CD3 molecules). This result indicates that the 14F7hT-2c11 antibody mediates lysis only in tumor cells such as P3X63 (Example 4). Figure 4 C), without mediating the lysis of normal cells (such as mouse T lymphocytes).

[0124] In the second experiment, total lymphocytes (potential target cells) from C57BL / 6 mouse lymph nodes were incubated with 14F7hT-UCHT1 (15 nM) for 72 hours in the presence of PBMCs from healthy human donors. Under these conditions, for each experimental condition, the number of live target cells (mouse lymphocytes) (LIVE / DEAD FixableNear-IRDeadcell- / mouse CD45-) was counted, and the percentage of live cells relative to the maximum viability condition used as a reference (mouse lymphocytes incubated with PBMCs without any treatment) was calculated. Each experimental condition was evaluated in triplicate.

[0125] like Figure 10As shown in B, mouse lymphocytes treated with 14F7hT-UCHT1 showed no statistically significant difference in viable cell percentage compared to mouse lymphocytes incubated with the negative control MOPC-UCHT1 (unrelated BAb-T, which does not recognize NGcGM3 gangliosides but does recognize human CD3 molecules, Zekri, L., et al. (2021) EMBOmolecular medicine 13(2): e11902) (Dunn post-hoc test, p<0.05). Similar to 14F7hT-2c11, this result indicates that BAb-T14F7hT-UCHT1 does not induce lysis of normal cells, despite its expression of the ganglioside, unlike tumor cells, which do mediate cytotoxic effects on tumor cells.

[0126] Example 9. The anti-NGcGM3 BAb-T showed no acute toxicity in vivo.

[0127] Once it was demonstrated that anti-NGcGM3 BAb-T cells did not lyse normal cells expressing the aforementioned gangliosides, we proceeded to evaluate their potential in vivo toxicity in a relevant animal model (mice) that naturally expresses NGcGM3 gangliosides (Ecsedy, JA, et al (1999) Journal of Neurochemistry 73(1): 254-259). For this purpose, a study was designed in which BALB / c nu / nu mice were intravenously injected with 20 μg of 14F7hT-UCHT1 on day 1 and intraperitoneally injected with 20 × 10⁻⁶ g of 14F7hT-UCHT1. 6 Personal PBMCs. This is a dose 10 times higher than the dose of BAb-T administered in this format in protocols used to evaluate antitumor efficacy (Zekri, L., et al. (2021) EMBO molecularmedicine 13(2):e11902; Mehta, NK, and cols (2022) Journal for immunotherapy of cancer 10(3):e003882). Additionally, groups receiving only PBMCs and groups receiving no treatment were included. Serum human IFN-γ levels were examined 24 hours later. Figure 11 A) No statistically significant differences were observed between the groups. After 48 hours, the mice were weighed and euthanized for subsequent processing of their organs.

[0128] like Figure 12As shown in A and B, treatment with BAb-T 14F7-UCHT1 and transfer to human PBMCs did not cause changes in animal body weight or relative organ weight, indicating that the antibody is virtually non-toxic even in animals expressing the gangliosides in their normal cells. Overall, these results suggest that co-application of 14F7hT-UCHT1 with human PBMCs does not induce unwanted T cell activation in the absence of tumors, and that the antibody is tolerable in animals with no evidence of acute toxicity.

[0129] In a parallel study, the potential toxic effects of the 14F7-2c11 antibody in vivo were evaluated. For this purpose, 14F7hT-2c11 antibody (50 μg) was administered intravenously, while another group received 145.2c11 antibody (a monospecific antibody against mouse CD3, the parent of 14F7hT-2c11, which induces T cell activation, Leo, O., et al. (1987) Proceedings of the National Academy of Sciences 84(5):1374-1378) via the same route (positive control).

[0130] like Figure 11 As seen in Figure B, serum IFN-γ levels measured 24 hours after BAb-T administration revealed that the 14F7hT-2c11 antibody did not induce the secretion of this cytokine in the presence of normal cells expressing NGcGM3. This secretion occurred only in mice treated with anti-mouse CD3 antibody (145.2c11). Additionally, mice were weighed daily during the study period. Figure 13 As shown, two days after administration, the group treated with the 145.2c11 antibody had a weight loss (g) relative to the control group (Tukey test, p<0.05), unlike the group treated with BAb-T.

[0131] These findings reinforce the evidence that anti-NGcGM3 BAb-T cells are activated only in the presence of tumors (whether in vitro or in vivo). Furthermore, they indicate minimal "targeted, detumescent" toxicity due to the absence of cytotoxicity to normal cells.

Claims

1. A bispecific antibody comprising an antibody, antibody fragment or scFv that recognizes NGcGM3 ganglioside on tumor cells and an antibody, antibody fragment or scFv that recognizes human CD3 molecules.

2. The bispecific antibody according to claim 1, wherein the antibody recognizing NGcGM3 is human IgG.

3. The bispecific antibody according to claim 1, wherein the antibody has a format selected from the group consisting of: TandAb, DART, DART-Fc, DuoBody, CrossMab, KiH, BiTE, Triomab, IgG-scFv.

4. The bispecific antibody according to claim 2, wherein the heavy chain is fused directly or via a peptide linker to a specific antibody, antibody fragment, or scFv targeting human CD3.

5. The bispecific antibody of claim 4, wherein the antibody, antibody fragment, or scFv recognizing the human CD3 molecule has a sequence selected from the group consisting of SEQ ID NO. 5-7 or variants of these sequences having more than 90% identity with said sequence.

6. The bispecific antibody of claim 4, wherein the antibody, antibody fragment or scFv recognizing NGcGM3 has a heavy chain variable region selected from the group consisting of SEQ ID NO. 10 and SEQ ID NO. 15 or variants of these sequences having more than 90% identity with these sequences.

7. The bispecific antibody according to claim 4, wherein the antibody, antibody fragment or scFv recognizing NGcGM3 has a light chain variable region selected from the group consisting of SEQ ID NO. 11-14 or variants of these sequences having more than 90% identity with them.

8. The bispecific antibody according to claim 2, wherein the human IgG sequence is selected from the group consisting of SEQ ID NO. 1-4 or variants of these sequences having more than 90% identity with them.

9. A pharmaceutical composition comprising a bispecific antibody as an active ingredient of any one of claims 1-8 and a pharmaceutically acceptable carrier.

10. Use of the bispecific antibody of any one of claims 1-8 in the treatment of lymphoproliferative disorders and solid tumors expressing NGcGM3.

11. Use of the nucleic acid encoding BAbs-T according to any one of claims 1-8 in gene therapy for treating tumors expressing NGcGM3.

12. The use according to claim 11, wherein the gene therapy is based on the injection of mRNA or transduction particles encoding BAbs-T.

13. A method of treating a subject in need of this treatment, comprising administering the bispecific antibody of any one of claims 1-8 in a dose range of 20 μg to 10 mg via subcutaneous, intravenous, intradermal, intramuscular, intratumoral, or intraperitoneal route.

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