Anti-idiotypic antibodies and uses thereof

Abstract

The present invention relates to an anti-idiotypic antibody, or an antigen-binding fragment thereof, which specifically binds to an idiotypic region of an anti-Trop-2 antibody or an antibody fragment of an anti-Trop-2 antibody. In a further embodiment, claimed herein is a method of detecting a CAR comprising a target antibody or antigen-binding fragment thereof. In a further embodiment, a method of inhibiting the toxicity of CAR T / NK cell therapy by using an anti-Id Ab according to the invention is described.

Description

Background Art

[0001] Methods are available for adoptive cell therapy using engineered cells expressing recombinant receptors, such as chimeric antigen receptors (CARs) containing extracellular antibody antigen binding domains.

[0002] CAR-T or CAR-NK cell therapy is a promising T / NK cell therapy engineering practice. This is based on artificial CARs that target specific tumor-associated antigens and directly link antibody antigen recognition with the cytotoxic activity of immune effector cells. Immune cells are isolated from the patient's peripheral blood (a procedure called leukapheresis), engineered in vitro to express a specific CAR, and once infused back into the patient, they trigger an immune response against the corresponding target antigen and cells expressing it.

[0003] The body's defense system can recognize molecules as self or non-self, including bacteria, viruses, and abnormal cancer cells. Cancer cells are recognized based on their antigenicity and immunogenicity acquired through the expression of antigens that are recognized as non-self. However, cancer cells can subvert the immune system's advantage, resulting in insufficient anti-tumor immunity and tumor survival and progression.

[0004] The creation of CARs has revolutionized T-cell-based immunotherapy for the treatment of certain cancers. CAR-T recognizes target antigens through the immunoglobulin antigen-binding region, thus bypassing the need for MHC (major histocompatibility complex) presentation.

[0005] CARs typically comprise an antibody fragment, such as a scFv or Fab fragment, incorporated into a fusion protein that also comprises other components, such as a CD3-ζ or CD28 transmembrane domain and a selective T cell activating portion, including the endodomain of CD3-ζ, CD28, OX40, 4-IBB, Lek and / or ICOS (Sadelain, Brentjens et al., 2013).

[0006] CAR constructs have also been used to direct the activity of natural killer (NK) cells (Hermanson and Kaufman 2015). NK cells are immune cells that recognize target cells in the absence of MHC and antibodies. As a result, they can trigger a rapid immune response (Klingemann 2014), which makes them important for destroying cells that mostly lack class I MHC.

[0007] Cancer cells that do not cause any inflammation are often regarded as self-cells by the immune system and cannot effectively stimulate T cell responses. NK cells produce several cytokines, including tumor necrosis factor α, interferon γ, and IL-10 (Jiang, Zhang et al., 2014). Activation of NK cells leads to the gradual formation of immune effector cells, such as dendritic cells, macrophages, and neutrophils, thereby promoting antigen-specific T cell and B cell responses.

[0008] NK cells do not require HLA matching; therefore, they can be used as allogeneic effector cells (Hermanson and Kaufman 2015). NK cells, unlike T cells, are easily redirected by CARs due to the lack of endogenous antigen specificity that targets the cells to be killed. The cell-targeting scFv or Fab can be connected to one or more intracellular signaling domains via a transmembrane domain to achieve lymphocyte activation. Signaling domains used with CAR-NK cells include CD3-ζ, CD28, 4-IBB, DAPIO, and OX40. NK cell lines such as NK-92, NKG, YT, NK-YS, HANK-I, YTS, and NKL have also been used for this purpose.

[0009] The main concern of CAR-T / NK therapy is the risk of a "cytokine storm" associated with a strong anti-tumor response mediated by a large number of activated T cells (Sadelain, Brentjens et al., 2013). This cytokine storm can cause fever, hypotension, hypoxia, neurological dysfunction and multiple organ failure.

[0010] Therefore, there is a fundamental need for the design of improved CAR constructs and CAR strategies to achieve better efficacy and reduce systemic toxicity, and to implement parallel approaches to reduce the risk of "cytokine storm" or other systemic toxicity.

[0011] Preventing these severe reactions may help mitigate on-target toxicity to non-tumor cells, where normal tissues expressing the target antigen are subject to toxicity caused by CAR-T or CAR-NK therapy. For example, severe transient inflammatory colitis was induced in all three patients with metastatic colorectal cancer who received CEA-targeted T cells (Parkhurst, Yang et al., 2011).

[0012] Therefore, there is an urgent need to control and manage CAR-T and CAR-NK cytotoxicity, especially when it reaches life-threatening levels. Different strategies have been tried to implement robust approaches to control CAR-T and CAR-NK cytotoxicity.

[0013] One of the oldest strategies to prevent these off-target effects is based on the introduction of suicide genes in CAR-T cells, which are being used in cancer immunotherapy (Jones, Lamb et al., 2014). Different genetically encoded molecules allow the selective death of genetically modified T cells to be induced by controlling metabolic pathways and dimerization induction. Such design leads to irreversible elimination of cells that cause uncontrolled toxicity and prevents damage to normal tissues. The two suicide genes currently verified are herpes simplex virus thymidine kinase (HSV-TK) and inducible caspase-9 (iCasp9) (Jones, Lamb et al., 2014).

[0014] Other strategies include regulating the activation of suicide genes to eliminate CAR-T cells while retaining a portion of cells that still have anti-tumor activity, and introducing inhibitory CARs to protect normal cells (Minagawa, Al-Obaidi et al., 2019).

[0015] The FDA recently approved tocilizumab, an anti-interleukin-6 receptor antagonist, for the treatment of CAR T cell-induced cytokine release syndrome in combination with glucocorticoids in refractory cases (Santomasso, Nastoupil et al., 2021).

[0016] However, these approaches are not satisfactory. They are mainly based on the introduction of additional genes. Many of these new molecules introduced into CAR-T cells and produced by CAR-T cells often induce immune responses in the host, thereby damaging CAR-T / NK cells.

[0017] In 1971, Niels Kaj Jerne modeled how the functional development of the immune system is formed during early ontogeny. In the thymus, lymphocytes that produce antibodies (Ab) against endogenous molecules are suppressed, resulting in the survival / selection of only "anti-non-self" antigens, which are recognized through a process of random mutation and clonal selection. This mechanism determines self-tolerance and a wide diversity of antibodies. Immunoglobulin genes are formed through V(D)J recombination, which joins the variable gene (V), diversity gene (D) and joining gene (J) germline genes. This results in each individual producing a different, unique range of antibody patterns, depending on the different histocompatibility antigens on the antigen-presenting cells and on random mutations involving the V genes.

[0018] The variable regions of T cell receptors (TCRs) and immunoglobulins contain complementary determining regions (CDRs) with unique amino acid structures that determine antigen specificity. The structure formed by each CDR is called a idiotype (Kieber-Emmons, Monzavi-Karbassi et al., 2012). The collection of idiotypes on a single antibody molecule determines the idiotype (Id) of the antibody (Jerne, 2004) ( Figure 1 ).

[0019] Immunization with anti-idiotypic (anti-Id) antibodies provides the impetus for anticancer immunotherapy approaches. According to the network hypothesis, idiotype-anti-idiotype interactions modulate the host immune response to a given antigen by inducing specific immune responses similar to those induced by the nominal antigen (Ag). Moreover, Id-based immunotherapy does not rely on vaccination with the original Ag or its fragments, precluding the possibility of concurrent undesirable side effects associated with traditional antigen vaccines. Moreover, the predictability of the fine specificity of tumor immune responses induced by anti-Id vaccines is higher than that of antigen vaccines.

[0020] The authors of the present invention designed and demonstrated the advantages of implementing a fully specific CAR-T / NK cell blocking method without any additional genetic modification. This was achieved by developing and utilizing anti-Trop-2 anti-Id antibodies, as described below.

[0021] Description of the invention

[0022] The present disclosure relates to anti-idiotypic antibodies (anti-Id Abs) that bind to or recognize anti-Trop-2 antibody portions, particularly anti-Trop-2 antibody portions present in recombinant receptors, including CARs.

[0023] The present disclosure also relates to the use of anti-Id Ab for specifically recognizing, detecting or selecting cells expressing the recombinant receptor, such as anti-Trop-2 CAR T cells or anti-Trop-2 CAR NK cells.

[0024] The present disclosure also relates to the use of anti-Id Abs for specific depletion of said cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 : Schematic representation of the effects caused by anti-Id Ab according to the present invention in the presence of anti-Trop-2 CAR-T cells. Idiotypic determinants are generated by the conformation of the amino acid sequences of the heavy and light chain variable regions that are specific for each antigen. Anti-Id Ab can recognize antigenic determinants in the variable regions of antibodies that are expressed in the extracellular part of CAR T cells.

[0026] Figure 2: Flow cytometry analysis of Jurkat cells transduced with anti-activated Trop-2 CAR. Horizontal axis: fluorescent staining. Vertical axis: cell forward scatter associated with cell size. Each dot represents the reading of a single cell. (A) Control of unstained cells. (B) Anti-FLAG-Alexa488 antibody binds to the synthetic tag Flag added to the CAR construct. (C) Anti-Hu2G10 idiotype (1D4-1 anti-Id Ab) followed by goat-anti-mouse Alexa488 fluorescent antibody to show binding. Ovals contain positive CAR expressing cells.

[0027] Figure 3 : ELISA assay shows the affinity of anti-Id Ab to CAR variable region. The wells of the ELISA plate were coated with 1D4-1 anti-Id Ab and incubated with serially diluted Hu2G10 mAb. Binding was detected using HRP-coupled goat-anti-human kappa polyclonal antibody. The absorbance values ​​(Y-axis) at each test antibody concentration (X-axis) are plotted in the figure. Background refers to the sample wells where all indicated procedures were performed but Hu2G10 was not applied. DETAILED DESCRIPTION OF THE INVENTION

[0029] Unless otherwise defined herein, all technical and scientific terms used have the same meanings as commonly understood by those skilled in the fields of gene therapy, biochemistry, genetics, and molecular biology.

[0030] Unless otherwise indicated, the practice of the present invention will employ conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA and immunology, which are within the technical scope of the art. Such techniques are fully explained in the literature. See, for example, Molecular Cloning: A Laboratory Manual, 3rd edition (Sambrook et al., 2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press).

[0031] Provided herein are reagents for binding or identifying antibodies and their antigen-binding fragments, including antibody fragments (e.g., scFv) and chimeric molecules (e.g., chimeric antigen receptors) containing them. Compositions and products containing such reagents are also provided, including those containing compositions and products of surfaces (e.g., solid surfaces, such as plates or beads) to which the reagents are bound. In addition, the embodiments provided herein also include the use and methods of using the reagents, compositions, and products, including for detecting, using, operating, and / or stimulating cells or therapies containing or suspected of containing the antibodies or chimeric molecules, such as for detecting, stimulating, or using cells expressed by CAR.

[0032] Trop-2, a hallmark of cancer cells and metastatic spread, was selected as a target for CAR engineering as an extremely specific means of controlling CAR-T and CAR-NK activity and potential side effects after treatment administration. Trop-2 targeted assays and therapeutic approaches known in the art rely on anti-Trop-2 mAbs that recognize a single immunodominant epitope located between the globular and stalk regions that is accessible and recognized by all cells expressing Trop-2.

[0033] In some embodiments, provided herein are anti-Id Abs or antigen-binding fragments thereof that bind to or recognize an anti-Trop-2 target antibody or antigen-binding fragment thereof.

[0034] In one embodiment, the anti-Trop-2 antibody is the 2G10 mAb described in WO2010089782.

[0035] In one embodiment, the anti-Trop-2 antibody is the humanized 2G10 mAb described in WO2016087651.

[0036] In one embodiment, the target is activated Trop-2, ie, a processed form of Trop-2 that is specifically and differentially expressed in tumor cells, wherein the processed form of Trop-2 is proteolytically cleaved between R87 and T88.

[0037] Antibodies against activated Trop-2 were generated using immunogens containing the entire extracellular portion of the Trop-2 molecule (SEQ ID NO: 1, corresponding to amino acids 31-274 of SEQ ID NO: 2) and individual domains (SEQ ID NO: 3, globular domain: amino acids 31-145 of SEQ ID NO: 1; SEQ ID NO: 4 "stem": amino acids 146-274 of SEQ ID NO: 2), wherein the activated Trop-2 is a Trop-2 molecule proteolytically cleaved between R87 and T88. These were produced in human 293 transformed renal epithelial cells and MCF-7 breast adenocarcinoma, mouse L fibrosarcoma and NS-0 myeloma, insect Sf9 and yeast cells in their native folded form. Expression vectors for production were generated using PCR amplification to fuse the Trop-2 coding sequence with tags for purification or to enhance immunogenicity. The PCR fragments were subcloned in the above vectors and expressed in the corresponding hosts. The Trop-2 protein was purified by affinity chromatography. According to the best procedures known in the art, BALB / c mice were subjected to multiple immunization cycles with the above-mentioned immunogens. According to methods known in the art, the spleen cells of the immunized mice were fused with Sp2 / 0 or NS-0 myeloma cells and the corresponding hybridomas were obtained. The antibodies produced by the hybridomas thus obtained were screened to determine their specificity and differential reactivity to processed Trop-2 expressed by tumor cells. mAbs 1A9 and 1B4 have been selected because of their ability to recognize and bind with high affinity only to tumor-specific processed forms of Trop-2 (activated Trop-2, wherein the activated Trop-2 is a Trop-2 molecule that is proteolytically cleaved between R87 and T88), rather than the unprocessed Trop-2 found in normal tissues. Flow cytometric cross-competition experiments between 1A9, 1B4 and T16 mAbs on KM12SM / wtTrop-2 and KM12SM / vector transfectants showed that 1A9 and 1B4 blocked the binding of each other, indicating that they recognize the same epitope. In contrast, there was no competition between 1A9 and 1B4 and T16, indicating that the procedure described herein allows efficient acquisition of mAbs directed against epitopes distinct from the immunodominant epitope.

[0038] In one embodiment, the anti-activated Trop-2 antibody is 1A9, which is secreted by a hybridoma deposited at the following International Depository Authority (IDA): Interlab Cell Line Collection, IRCCS Ospedale Policlinico San Martino, Genoa, Italy, and assigned the deposit number PD21006.

[0039] In one embodiment, the anti-activated Trop-2 antibody is 1B4, which is secreted by a hybridoma deposited at the following International Depository Authority (IDA): Interlab Cell Line Collection, IRCCS Ospedale Policlinico San Martino, Genoa, Italy, and assigned the deposit number PD21005.

[0040] C57BL / 6 have been immunized with humanized 2G10 antibody and then stimulated with murine 2G10 antibody. Hybridomas have been obtained, and the preferred hybridoma is 1D4-1 anti-Id Ab.

[0041] In a preferred embodiment, the 1D4-1 anti-Id Ab is secreted by a hybridoma deposited at the following International Depository Authority (IDA): Interlab Cell Line Collection, IRCCS Ospedale Policlinico San Martino, Genoa, Italy, and assigned the deposit number PD21004.

[0042] In some of any such embodiments, the anti-Id Ab or antigen-binding fragment specifically binds to an anti-Trop-2 target antibody or antigen-binding fragment contained within an antigen binding domain included in the extracellular portion of the CAR.

[0043] In some of any such embodiments, scFv is within or included in the extracellular portion of CAR. In some of any such embodiments, the anti-Id Ab or antigen binding fragment binds to the scFv contained in or included in the extracellular portion of CAR. In some of any such embodiments, the CAR also includes a transmembrane domain connected to the antigen binding domain by a spacer. In some of any such embodiments, the spacer is an immunoglobulin spacer. In some of any such embodiments, the spacer includes an amino acid motif GlyGlyGlyGlySer, and more preferably, the motif is repeated 4 times. In some of any such embodiments, the transmembrane domain includes a transmembrane portion of CD28. In some of any such embodiments, the transmembrane portion of CD28 is human CD28. In some of any such embodiments, the anti-Id Ab or its antigen binding fragment does not bind to an epitope in the spacer domain of CAR.

[0044] In some of any such embodiments, the anti-Id Ab or antigen-binding fragment thereof does not bind to an epitope in an Fc domain. In some of any such embodiments, the Fc domain is a human IgG1 Fc domain.

[0045] In some of any such embodiments, the anti-Id Ab or its antigen binding fragment is an agonist of a CAR comprising an anti-Trop-2 target antibody or its antigen binding fragment. In some of any such embodiments, the anti-Id Ab or its antigen binding fragment is an agonist of a CAR in a soluble form. In some of any such embodiments, the anti-IdAb or its antigen binding fragment is an agonist of a CAR when fixed to a support or a stationary phase. In some of any such embodiments, the support or stationary phase is a plate or a bead.

[0046] In some of any such embodiments, the binding affinity (Kd) of the anti-Id Ab to the anti-Trop-2 target antibody or antigen-binding fragment thereof is or is about or is less than or is about 100 nM, 50 nM, 40 nM, 30 nM, 25 nM, 20 nM, 19 nM, 18 nM, 17 nM, 16 nM, 15 nM, 14 nM, 13 nM, 12 nM, 11 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM or 0.1 nM or 0.01 nM or 0.001 nM.

[0047] In some of any such embodiments, the anti-Id Ab or antigen-binding fragment thereof is an intact antibody or a full-length antibody.

[0048] In some embodiments, a conjugate is also provided herein, comprising an anti-Id Ab or its antigen-binding fragment and a heterologous molecule or portion in any embodiment provided herein. In some of any such embodiments, the heterologous molecule or portion is a label. In some of any such embodiments, the label is selected from fluorescent dyes, fluorescent proteins, radioisotopes, chromophores, metal ions, gold particles, silver particles, magnetic particles, polypeptides, enzymes, streptavidin, biotin, luminescent compounds and oligonucleotides. In some of any such embodiments, the heterologous molecule or portion is a protein, peptide, nucleic acid or small molecule, which is optionally or comprises a toxin or Strep-Tag.

[0049] In some embodiments, provided herein is also a composition comprising the anti-Id Ab or antigen-binding fragment thereof of any such embodiments or a conjugate of any such embodiments.

[0050] In some of any such embodiments, the composition further comprises a pharmaceutically acceptable excipient.

[0051] In some of any such embodiments, the composition further comprises a compound active in the treatment of cancer.

[0052] In some embodiments, also provided herein is a kit comprising one or more anti-Id Abs or antigen-binding fragments thereof of any such embodiments, a conjugate of any such embodiments, and optionally instructions for use.

[0053] In some embodiments, the present invention also provides a method for detecting a CAR comprising a target antibody or an antigen-binding fragment thereof, comprising:

[0054] (a) contacting a cell expressing a CAR comprising a target antibody or antigen-binding fragment thereof with an anti-Id Ab or antigen-binding fragment thereof of any of such embodiments that specifically binds to the target antibody or antigen-binding fragment thereof, or a conjugate of any of such embodiments; and

[0055] (b) detecting cells bound to the anti-Id Ab or the antigen-binding fragment thereof.

[0056] In some of any such embodiments, the anti-Id Ab or antigen-binding fragment thereof is directly or indirectly labeled for detection.

[0057] In some embodiments, the present invention also provides a method for selecting a cell from a cell population, comprising:

[0058] (a) contacting a population of cells expressing a CAR comprising a target antibody or antigen-binding fragment thereof, or a cell that binds to a target antibody or antigen-binding fragment thereof, with an anti-Id Ab or antigen-binding fragment thereof of any such embodiment, or a conjugate of any such embodiment, which specifically binds to the target antibody or antigen-binding fragment thereof; and

[0059] (b) selecting cells that bind to the anti-Id Ab or antigen-binding fragment thereof. In some of any such embodiments, cells that bind to the anti-Id Ab or antigen-binding fragment thereof are selected by affinity-based separation.

[0060] In some of any such embodiments, the affinity-based separation is an immunoaffinity-based separation. In some of any such embodiments, the affinity-based separation is performed by flow cytometry. In some of any such embodiments, the affinity-based separation is performed by magnetic activated cell sorting. In some of any such embodiments, the affinity-based separation comprises affinity chromatography. In some of any such embodiments, the anti-idiotypic antibody or antigen-binding fragment thereof is reversibly bound or fixed to a support or stationary phase.

[0061] In some embodiments, there is also provided herein a method of depleting cells, the method comprising administering a composition to a subject in need, the composition comprising any of such embodiments of anti-Id Ab or its antigen-binding fragment, or any of such embodiments of the conjugate, which specifically binds to the target antibody or its antigen-binding fragment, wherein the subject has administered a cell expressing a CAR comprising the target antibody or its antigen-binding fragment. In some of any such embodiments, the depletion occurs by antibody-dependent cell-mediated cytotoxicity (ADCC).

[0062] In one embodiment, the pharmaceutical composition claimed herein comprises an anti-Id Ab, preferably, the anti-IdAb is 1D4-1 Ab.

[0063] In one embodiment, the pharmaceutical composition further comprises a plurality of anti-Trop-2 CAR engineered cells.

[0064] In one embodiment, the pharmaceutical composition is claimed herein for use in a method of treating a disease in a subject. In one embodiment, the disease is a tumor.

[0065] In one embodiment, the anti-Id Ab inhibits the strength of the anti-Trop-2 CAR cell based therapy.

[0066] The authors of the present invention surprisingly demonstrated that the anti-Id Ab according to the present invention inhibited the toxicity of anti-Trop-2 CAR-T / NK cells.

[0067] The following are examples that illustrate methods of practicing the present invention. These examples should not be construed as limiting. Example

[0068] Embodiment 1:

[0069] Jurkat cells were transduced with an anti-activated Trop-2 CAR, wherein the VH had a protein sequence as shown in SEQ ID NO: 5, a DNA sequence as shown in SEQ ID NO: 6, and the VL had a protein sequence as shown in SEQ ID NO: 7, a DNA sequence as shown in SEQ ID NO: 8. Figure 2 B) or anti-Hu2G10 idiotype (1D4-1 anti-IdAb), followed by goat anti-mouse Alexa488 fluorescent antibody staining ( Figure 2 C), and then the cells were analyzed by FACS. Unstained cells were also analyzed as a control ( Figure 2 A). CAR element shows fluorescent staining. That is, CAR is expressed at the membrane level and can bind to 1D4-1 anti-Id Ab.

[0070] SEQ ID NO:5

[0071] QVQLVQSGAEVKKPGASVKVSCKASGFTFSSSYISWLRQAPGQRLEWIAWIYAGTGGTSYNQKFTGKATLTVDTSASTAYMELSSLRSEDTAVYYCARHNPRYYAMDYWGQGTTVTVSS

[0072] SEQ ID NO:6

[0073] CAAGTGCAGCTCGTCCAGTCTGGAGCTGAAGTCAAAAAGCCTGGGGCTTCAGTGAAAGTCTCCTGCAAGGCTTCTGGCTTCACCTTCAGCAGTAGCTATATCAGTTGGTTGAGGCAGGCCCCTGGACAGAGACTTGAGTGGATTGCATGGATTTATGCTGGAACTGGCGGAACTAGCTATAATCAGAAGTTCACAGGCAAGGCCACACTGACTGTAGACACATCCGCCAGCACAGCCTACATGGAACTCAGCAGCCTGAGATCTGAGGACACTGCCGTCTATTACTGTGCAAGACATAACCCTCGTTACTATGCTATGGACTACTGGGGCCAAGGAACCACAGTCACCGTCTCCTCA

[0074] SEQ ID NO:7

[0075] DTQMTQSPSSLSASVGDRVTITCITSTDIDDDMNWYQQKPGKAPKLLISEGNTLRPGVPSRFSGSGYGTDFTFTISSLQPEDIATYYCLQSDNLPYTFGGGTKVEIKR

[0076] SEQ ID NO:8

[0077] GACACCCAGATGACCCAGTCTCCAAGCTCCCTGTCCGCCAGCGTGGGAGATAGAGTCACCATCACATGCATCACCAGCACTGATATTGATGATGATATGAACTGGTACCAGCAGAAGCCAGGGAAAGTCCCTAAGCTCCTGATTTCAGAAGGCAATACTCTG CGCCCTGGAGTCCCATCCCGATTCTCCGGCAGTGGCTATGGAACAGATTTTACCTTTACAATTAGCTCCCTGCAGCCAGAAGATATTGCAACCTACTACTGTTTGCAAAGTGATAACCTGCCCTACACCTTCGGAGGGGGGACCAAAGTCGAAATCAAACGG

[0078] Embodiment 2:

[0079] The wells of the ELISA plate were coated overnight at 4°C with 1 μg / ml 1D4-1 Ab (lot 9 / 26 / 14) in 100 μl / well PBS. After blocking with SuperBlock (Thermo Fisher Scientific), Hu2G10 diluted to 1 μg / ml in SuperBlock spiked with 2% human serum was applied and serially diluted 3-fold in SuperBlock containing 2% serum. Binding was detected using an HRP-conjugated goat anti-human kappa polyclonal antibody in SuperBlock. The absorbance value (Y-axis) was calculated at each test antibody concentration (X-axis). Background was calculated in sample wells where all indicated procedures were performed but Hu2G10 was not applied.

[0080] References

[0081] Hermanson, DL and DS Kaufman (2015). "Utilizing chimericantigenreceptors to direct natural killer cell activity." Front Immunol 6:195.

[0082] Jerne,NK(2004)."The somatic generation of immune recognition.1971."Eur J Immunol 34(5):1234-1242.

[0083] Jiang, H., W. Zhang, P. Shang, H. Zhang, W. Fu, F. Ye, T. Zeng, H. Huang, X. Zhang, W. Sun, D. Man-Yuen Sze, Q. Yi and J. Hou (2014). "Transfection of chimeric anti-CD138 gene enhances natural killer cell activation and killing of multiple myeloma cells." Mol Oncol 8(2):297-310.

[0084] Jones, B. S., L. S. Lamb, F. Goldman and A. Di Stasi (2014). "Improving the safety of cell therapy products by suicide gene transfer." Front Pharmacol 5:254.

[0085] Kieber-Emmons, T., B. Monzavi-Karbassi, A. Pashov, S. Saha, R. Murali and H. Kohler (2012). "The promise of the anti-idiotype concept." Front Oncol 2:196.

[0086] Klingemann, H. (2014). "Are natural killer cells superior CAR drivers?" Oncoimmunology 3:e28147.

[0087] Minagawa, K., M. Al-Obaidi and A. Di Stasi (2019). "Generation of Suicide Gene-Modified Chimeric Antigen Receptor-Redirected T-Cells for Cancer Immunotherapy." Methods Mol Biol 1895:57-73.

[0088] Parkhurst, M.R., J.C. Yang, R.C. Langan, M.E. Dudley, D.A. Nathan, S.A. Feldman, J.L. Davis, R.A. Morgan, M.J. Merino, R.M. Sherry, M.S. Hughes, U.S. Kammula, G.Q. Phan, R.M. Lim, S.A. Wank, N.P. Restifo, P.F. Robbins, C.M. Laurencot and S.A. Rosenberg (2011). "T cells targeting carcinoembryonic antigen can mediate regression of metastatic colorectal cancer but induce severe transient colitis." Mol Ther 19(3):620 - 626.

[0089] Sadelain, M., R. Brentjens and I. Riviere (2013). "The basic principles of chimeric antigen receptor design." Cancer Discov 3(4):388 - 398.

[0090] Santomasso, B.D., L.J. Nastoupil, S. Adkins, C. Lacchetti, B.J. Schneider, M. Anadkat, M.B. Atkins, K.J. Brassil, J.M. Caterino, I. Chau, M.J. Davies, M.S. Ernstoff, L. Fecher, P. Funchain, I. Jaiyesimi, J.S. Mammen, J. Naidoo, A. Naing, T. Phillips, L.D. Porter, C.A. Reichner, C. Seigel, J.-M. Song, A. Spira, M. Suarez-Almazor, U. Swami, J.A. Thompson, P. Vikas, Y. Wang, J.S. Weber, K. Bollin and M. Ghosh (2021). "Management of Immune-Related Adverse Events in Patients Treated With Chimeric Antigen Receptor T-Cell Therapy: ASCO Guideline." Journal of Clinical Oncology 39(35): 3978-3992.

[0091]

[0092]

Claims

1. An anti-idiotypic antibody (anti-Id Ab) or an antigen-binding fragment thereof, wherein the anti-Id Ab or the antigen-binding fragment thereof specifically binds to the idiotypic region of an anti-Trop-2 antibody or an antibody fragment of an anti-Trop-2 antibody.

2. The anti-Id Ab according to claim 1, which is an isolated monoclonal 1D4-1 anti-Id Ab, which is produced by a hybridoma cell line deposited in the following International Depository Authority (IDA): Interlab Cell Line Collection Center, IRCCSOspedale Policlinico San Martino, Genoa, Italy, and assigned the deposit number PD21004.

3. The anti-Id Ab according to claim 1, wherein the anti-Trop-2 antibody or antibody fragment specifically recognizes activated Trop-2, wherein the activated Trop-2 is a Trop-2 molecule proteolytically cleaved between R87 and T88.

4. The anti-Id Ab according to claim 1, which binds to an anti-Trop2 target antibody or antigen-binding fragment contained in or included in the antigen-binding domain of the extracellular portion of CAR.

5. A pharmaceutical composition comprising the anti-Id Ab according to any one of claims 1-4.

6. The composition of claim 5, further comprising one or more other compounds effective in the treatment of cancer.

7. The composition of claim 5 or 6, further comprising a plurality of anti-Trop-2 CAR engineered cells.

8. A method for detecting a CAR comprising a target antibody or an antigen-binding fragment thereof, the method comprising: (a) contacting a cell expressing a CAR comprising a target antibody or an antigen-binding fragment thereof with an anti-Id Ab or an antigen-binding fragment thereof according to any one of claims 1 to 4, or a conjugate of any one of them, which specifically binds to the target antibody or an antigen-binding fragment thereof; and (b) detecting cells bound to the anti-Id Ab or the antigen-binding fragment thereof.

9. A method for selecting a cell expressing CAR, the method comprising: (a) contacting a cell population expressing a CAR comprising a target antibody or an antigen-binding fragment thereof or a cell bound to a target antibody or an antigen-binding fragment thereof with an anti-Id Ab or an antigen-binding fragment thereof or a conjugate that specifically binds to a target antibody or an antigen-binding fragment thereof according to any one of claims 1 to 4; and (b) selecting cells that bind to the anti-Id Ab or antigen-binding fragment thereof.

10. The method for detecting a CAR comprising a target antibody or an antigen-binding fragment thereof according to claim 8, wherein: In the target antibody or antigen-binding fragment, VH has a protein sequence as shown in SEQ ID NO:5 and a DNA sequence as shown in SEQ ID NO:6, and VL has a protein sequence as shown in SEQ ID NO:7 and a DNA sequence as shown in SEQ ID NO:

8.

11. The method of claim 10, wherein the anti-Id A is 1D4-1.

12. The method for selecting a cell expressing CAR according to claim 9, wherein: In the target antibody or antigen-binding fragment, VH has a protein sequence as shown in SEQ ID NO:5 and a DNA sequence as shown in SEQ ID NO:6, and VL has a protein sequence as shown in SEQ ID NO:7 and a DNA sequence as shown in SEQ ID NO:

8.

13. The method of claim 12, wherein the anti-Id A is 1D4-1.

14. A composition according to claim 6 or 7 for use in a method of treating a disease in a subject.

15. The composition for use according to claim 14, wherein the disease is a solid tumor expressing TROP-2.

16. The composition for use according to claim 14, wherein use of the composition comprises administering a plurality of cells expressing a CAR comprising a target antibody or an antigen-binding fragment thereof prior to or in combination with administering the composition.

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