WO2024127444A1 - Cellules effectrices de récepteur antigénique chimérique (car b7-h3) spécifiques de b7-h3 dérivées d'igm pour le traitement de tumeurs cd276+ et de maladies auto-immunes - Google Patents

Cellules effectrices de récepteur antigénique chimérique (car b7-h3) spécifiques de b7-h3 dérivées d'igm pour le traitement de tumeurs cd276+ et de maladies auto-immunes Download PDF

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WO2024127444A1
WO2024127444A1 PCT/IT2023/095002 IT2023095002W WO2024127444A1 WO 2024127444 A1 WO2024127444 A1 WO 2024127444A1 IT 2023095002 W IT2023095002 W IT 2023095002W WO 2024127444 A1 WO2024127444 A1 WO 2024127444A1
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cells
car
sequence
antigen receptor
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Concetta QUINTARELLI
Biagio DE ANGELIS
Ignazio CARUANA
Lorenzo Moretta
Cristina Bottino
Roberta Castriconi
Franco LOCATELLI
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Ospedale Pediatrico Bambino Gesu'
Universita' Degli Studi Di Genova
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464411—Immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/10—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the structure of the chimeric antigen receptor [CAR]
    • A61K2239/22—Intracellular domain
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/10—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the structure of the chimeric antigen receptor [CAR]
    • A61K2239/23—On/off switch
    • A61K2239/25—Suicide switch
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/39—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by a specific adjuvant, e.g. cytokines or CpG
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/47—Brain; Nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/48—Blood cells, e.g. leukemia or lymphoma
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/54—Pancreas
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)

Definitions

  • B7-H3 CAR IgM derived B7-H3-Specific Chimeric Antigen Receptor
  • the present invention concerns IgM derived B7-H3-Specific Chimeric Antigen Receptor (B7-H3 CAR) effector cells for the treatment of B7-H3 (CD276) positive tumors and autoimmune diseases.
  • B7-H3 CAR IgM derived B7-H3-Specific Chimeric Antigen Receptor
  • the present invention concerns a vector including the cassette coding for B7-H3 CAR gene obtained using the single chains variable fragments (scFv) of the monoclonal IgM antibody M5B14, a method for the production thereof and B7-H3 CAR genetically modified effector cells (such as T cells or innate cells such as NK and NK-T cells) for the treatment of B7-H3 (CD276) positive tumors, such as lymphoid malignancies, leukemia and solid tumors such as CNS tumors, extracranial and intracranial tumors, and autoimmune diseases.
  • scFv single chains variable fragments
  • B7-H3 [B7-homolog 3 or CD276] is a type I transmembrane protein encoded by chromosome 15 in humans (PMID: 27208063).
  • B7-H3 is both an inhibitory ligand for natural killer cells and T cells and a tumor antigen widely expressed among human solid tumours (PMID 26487718).
  • B7-H3 which belongs to the B7/CD28 superfamily, is constitutively found at low level also on non-immune resting cell types, fibroblasts, endothelial cells (EC), osteoblasts, and amniotic fluid stem cells. Moreover, B7-H3 expression is induced on immune cells, specifically APCs (PMID: 17615586). In addition, B7-H3 can also be detected on natural killer (NK) cells, B cells, and a minor population of T cells following PMA/ionomycin stimulation. (PMID: 14764704).
  • NK natural killer
  • B7-H3 is found on the membrane, in the cytoplasm, or within the nucleus of cancer cells, but also on the tumor-associated vasculature (PMID: 22473715). Recently, several studies have demonstrated aberrant overexpression of B7- H3 on tumor cells and cancer-associated stromal cells in both solid and hematologic malignancies including, leukaemia.
  • B7-H3 protein has been found overexpressed in acute myeloid leukemia (PMID: 26376842), prostate cancer (PMID: 17686830), breast cancers (PMID: 21107115) (PMID: 29344150), melanoma (PMID: 21671471 ), colorectal cancer (PMID: 31466914) (PMID: 22473715), ovarian cancer (PMID: 28765941 ) neuroblastoma (PMID: 15314238), osteosarcoma (PMID: 23940627), and Central Nervous System (CNS) tumors (PMID: 31887631 ).
  • B7-H3 expression was detected also on tumor cells lacking the G2D tumor associated marker, associated to resistence to the immunotherapeutic protocol based on the infusion of a anti-G2D mAb (PMID: 33795387).
  • the expression of B7-H3 was upregulated by IFN-y on neuroblastoma cells, particularly when cultured in 3D condition (PMID: 31447858, 35205760).
  • B7-H3-specific monoclonal antibodies (mAbs) and antibody-drug conjugates showed antitumor activity against B7-H3+ tumor cells in xenograft mouse models; and several phase I clinical trials showed a good safety profile (PMID: 33051306.)
  • Immunotherapeutic approaches targeting B7-H3 by CAR has been demonstrated of value in preclinical solid tumors and brain tumors models (PMID: 30655315) (PMID: 32341579) (PMID: 31466914) (PMID: 30753824) (PMID: 33653946), in acute leukemia (PMID: 33531429) and Anaplastic Large Cell lymphomas (PMID: 33348781 ).
  • Table 1 summarizes B7-H3 CAR T-cells characterized in preclinical models (Table 1 ).
  • the symbols used in this table are mAb: monoclonal antibodies; ScFv: single chain variable fragment.
  • GFP Green Fluorescence Protein; mCherry is a member of the mFruits family of monomeric red fluorescent proteins (mRFPs).
  • tdTomato is a basic (constitutively fluorescent) orange fluorescent protein.
  • Second-generation B7-H3 CAR T-cells whose binder is derived from a humanized antibody (Ab) (MGA271 , enoblituzumab) were developed and published in 2019 (PMID: 30655315).
  • the B7-H3 single chain variable fragment (scFv) was introduced into an MSGV.1 retroviral expression vector containing a CD8-a hinge- transmembrane domain, a CD137 (4-1 BB) costimulatory motif, and a CD3z signaling domain (second generation of CAR).
  • the sequence of the human IgG 1 constant domain CH2-CH3 was inserted between the scFv and the transmembrane (TM) domains.
  • B7-H3 CAR T cells were growth in media containing interleukin 2 (IL-2).
  • IL-2 interleukin 2
  • Preclinical studies demonstrated the ability of these cells to lyse orthotopic models of osteosarcoma (MG63.3 cell line), Ewing sarcoma (EW8 cell line), MB (DAOY and D425 cell line) and Atypical teratoid/rhabdoid tumors (ATRTs) xenograft (PMID: 32341579).
  • B7-H3.CAR-Ts second-generation B7-H3 CAR T-cells
  • scFv single-chain variable fragment derived from the B7-H3 376.96 mAb, (PMID: 24216048) (PMID: 6951087) (PMID: 28104527) and including either CD28 or 4-1 BB endodomains (B7-H3. CAR-28 ⁇ and B7-H3.CAR-BB , respectively).
  • Second- generation B7-H3.CAR-Ts contained central-memory, effector-memory, and T stem cell memory, without significant differences between CD28 and 4-1 BB costimulation.
  • both 376.96-B7-H3.CAR-Ts showed to be effective also against GBM cell lines and patient-derived GBM neurospheres in vitro and in xenograft murine models. No significant differences were found between CD28 and 4-1 BB co-stimulation, although CD28-co-stimulated CAR-T cells released more inflammatory cytokines (PMID: 31466914).
  • B7-H3.CAR-Ts exhibit efficient antigen-dependent cytotoxicity in vitro and in xenograft models of AML, and are unlikely to cause unacceptable hematopoietic toxicity. (PMID: 33531429)
  • a similar B7-H3.CAR lentiviral vector was proposed including the same single-chain variable fragment (scFv) derived from the published humanized 8H9 mAb (PMID: 26487718), CD28 and 4-1 BB as costimulatory endodomains, and the Green Fluorescence Protein (GFP) as trackable marker (PMID: 33811153).
  • scFv single-chain variable fragment
  • CD28 and 4-1 BB costimulatory endodomains
  • GFP Green Fluorescence Protein
  • TNBC triplenegative breast cancer
  • HNSCC head and neck squamous cell carcinoma
  • NSCLC non-small cell lung carcinoma
  • M21 skin cutaneous melanoma SKCM cell line
  • B7-H3-redirected CAR was based on a lentiviral vector encoding the B7-H3 binder J42-scFv, CD28 and 4-1 BB costimulatory domains, and the CD3- z signaling domain; mCherry was inserted as a tracker for detecting the expression of CAR with FACS.
  • B7-H3-targeted CAR-T cells exhibit antitumor effects on hematologic tumors (B-myelomonocytic leukemia MV-4-11 , histiocytic lymphoma U937), and solid tumors: human melanoma cell line A375, and hepatocellular carcinoma HepG2. (PMID: 32346608)
  • lentiviral vector containing a TanCAR molecule consisting of a CD8 leader, followed by CD70 specific scFv that is separated from B7-H3 specific scFv (clone: mAb-J42) by a 15- amino acid glycine/ serine repeat linker, hinge domain, CD8 transmembrane, the signaling domain of the costimulatory molecule 4-1 BB, the signaling domain of the T cell receptor CD3-zeta chain.
  • a P2A ribosome skip sequence separates the CAR sequence from a tdTomato as a CAR-T cell tracker. (PMID: 32685008).
  • this bivalent targeting CAR-T cell could not only induce a more superior antitumor effect but also induces regression of tumor in a lower dose than unspecific CAR-T cells (PMID: 32685008).
  • Second lentiviral vectors B7-H3-CARs utilizing a single-chain variable fragment (scFv) derived from the humanized B7-H3-specific monoclonal antibody (mAb) MGA271 , with different hinge/transmembrane (CD8a versus CD28) and CD28 or 41 BB costimulatory domains (CD8a/CD28, CD8a/41 BB, CD28/CD28, CD28/41 BB) has been evaluated in vitro and in vivo xenograft models.
  • CD8a/CD28-CAR T cells consistently outperformed other CAR T cell populations in three animal models, resulting in a significant survival advantage.
  • the trackable markers mostly used in known B7-H3.CARs, GFP, mCherry and tdTomato are intracellular reporter molecules, to evaluate gene transfer and expression, which can be detected in living cells without selection or staining.
  • B7H3-specific chimeric antigen receptors (B7-H3.CAR) of third generation are now provided.
  • bicistronic vectors have been designed, which allow the simultaneous expression of two transgenes, namely the inducible Caspase 9 (iC9) and the third generation B7-H3.CARs.
  • a clonal retroviral producer cell line has been generated that is able to produce high titer of retroviral vector containing: a ACD34 flag, represented by the extracellular domain of human CD34 linked to the CD8 transmembrane portion, with a double function: a)the selection of the genetically modified cells by clinical grade microbeads; b)the phenotypic identification of the genetically modified cells.
  • the CAR construct was cloned after the gene cassette including the sequence of iC9 using a 2A sequence.
  • the vectors mentioned above comprise or consist of: an inducible Caspase 9 (iC9) suicide gene as safety switch (PMID: 25389405; PMID: 29872565);
  • T2A self-cleaving peptides sequence, which can induce ribosomal skipping during translation of a protein in a cell
  • a signal peptide a single chain variable fragment (scFv) from (IgG) NE97 hybridoma, which was never applied for CAR therapy before; or an alternative single chain variable fragment (scFv) from (IgM) hybridoma, which was never applied for CAR therapy before; a trackable marker CD34 derived epitope (ACD34) of only 16 amino acid (aa) (as trackable marker) for a rapid identification by FACS (Fluorescence-activated cell sorting) System of gene modified T cells; - an hinge represented by CD8 regions to avoid the immunogenic CH2-CH3 murine sequence (PMID: 25212991 ); a transmembrane domain from the transmembrane domain of CD8 (CD8tm) to improve molecule stabilization; a link domain (of only 7 aa
  • Table 2 shows the peculiar elements that are present in the B7-H3.CARs according to the present invention in comparison with known B7-H3.CAR, reported in table 1.
  • the symbols used in this table are IgM: Immunoglobulin M; IgG: Immunoglobulin G; IC9: inducible Caspase 9; ACD34: CD34 derived epitope.
  • the CAR molecules according to the present invention comprise two different single chains variable fragments (scFv): a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of immunoglobulins, connected with a short linker peptide of ten to about 25 amino acids.
  • the two scFv were derived from the monoclonal IgG antibody NE97 or from the monoclonal IgM antibody M5B14, respectively.
  • the scFv are cloned in frame with CD8 transmembrane domain, CD284.1 BB or CD28.OX40 costimulatory domains, and CD3 zeta (CD3 cytoplasmic domain for the transduction of the activator signal after antigen engagement.
  • the clinical grade CAR construct was cloned in a retroviral vector after the gene cassette including the sequence of iC9 through the use of a 2A sequence.
  • Table 3 shows the functional elements of NE97.B7-H3.CARs vectors.
  • Retroviral encapsidation signal (psi; for packaging of RNA into virion particles);
  • iC9 gene contains the intracellular portion of the human caspase 9 protein, a pro-apoptotic molecule, fused to a drug-binding domain derived from human FK506-binding protein (FKBP12.caspase9, (iC9));
  • - 2A - encodes a synthetic 18 amino acid peptide from Thosea Asigna insect virus, which functions as a cleavable linker between the iC9 protein and CAR proteins;
  • a trackable marker CD34 derived epitope of only 16 amino acid (aa) (as trackable marker) for a rapid identification by FACS (Fluorescence- activated cell sorting) System and/or selection by Cell Sorter System of gene modified cells;
  • CD8 cytoplasmatic portion between the CD8TM and intracellular domains CD28.4-1BB-CD3 chain (4.1 BB- ; - 3’ LTR - Retroviral long terminal repeat at 3’ end of vector (functions as terminator/ polyadenylation sequences).
  • Table 4 shows the functional elements of M5B14.B7-H3.CARs vectors.
  • Results have been compared with those obtained using (NE97) B7-H3.CAR, including the scFv of the NE97 IgG anti-B7-H3 mAb.
  • (NE97) B7-H3 shows advantages in comparison with the (M5B14) B7-H3.CAR, in term of more efficient and stable CAR expression on T cells, a longer in vivo persistence, and higher antitumor activities thanks to the affinity of the NE97 scFv with the antigen.
  • the (M5B14) B7-H3.CAR with anti-B7-H3 single-chain variable fragment (scFv) derived from a murine antibody of IgM class is equally effective to (NE97)B7- H3.CAR in several solid and hematological tumor (as show in Figure 5A, 5C; 6E-H).
  • known CARs comprise single-chain variable fragment (scFv) derived usually from a murine/human antibody of IgG class, not from a murine antibody/human of IgM class.
  • IgM has low affinity but high avidity for the cognate antigen because it has 10 weak binding sites for antigen as opposed to the 2 binding sites of IgG with higher single binding affinities.
  • CAR configuration for both M5B14 and NE97 there are only 2 binding sites, that is, the variable region of the light (VL) and heavy chain (VH) of the single chain.
  • VL variable region of the light
  • VH heavy chain
  • the ideal CAR target antigen would be a native, surface- exposed tumor neo-antigen that is highly expressed and is undetectable in healthy tissues.
  • many commonly targeted solid tumor antigens are also expressed by non-tumor tissues, albeit at lower levels.
  • CAR molecules with high affinity to such antigens can lead to collateral targeting of healthy tissues resulting in on-target, off-tumor toxicity, a major limiting factor to the progress of CAR T cell therapy to date (PMID: 29085043).
  • B7-H3.CAR based on IgM class could be safer. Therefore (M5B14) B7-H3.CAR could be less toxic.
  • GTMP Gene therapy medicinal products
  • modified polyclonal (NE97) B7-H3.CAR T cells according to the present invention were able to eliminate very efficiently, in long-term co-culture, B7- H3+ tumours.
  • the biological products according to the present invention in xenograft in vivo models show to eliminate both paediatric and adult haematological solid tumors (including the tumor of SNC), the rhabdomyosarcoma tumor cells, colorectal tumor, the glioma brain tumor and to establish a long-term immunological memory.
  • the supernatants obtained by all SFG retroviral vector were able to transduce efficiently activated T cells, with very high level of transduction.
  • the introduction in both construct of CD34 derived epitope as trackable marker let easily to track the genetically modified T cells (CD3+CD34+) in vitro and in vivo xenograft mouse models.
  • NE97.B7-H3.CAR T-cells showed a superior anti-tumor activity against some solid tumors, such as the neuroblastoma cell lines SHSY5Y ( Figure 6A) and IMR-32 ( Figure 6B), the Ewing sarcoma A673 ( Figure 6C) and the ERMS cell line RD ( Figure 6D), compared to NT T-cells and M5B14.B7-H3.CAR T- cells, all four different clinical grade third generation of B7-H3.CAR T-cells according to the present invention are very active against B7-H3+leukemias/lymphomas cell lines and solid tumor cell lines.
  • B7-H3+leukemias/lymphomas cell lines such as the Hodgkin lymphoma cell line HDML-2 ( Figure 6E), the acute myeloid leukemia cell line OCI- AML3 ( Figure 6F), the Acute monoblastic/monocytic leukemia cell line MV4-11 ( Figure 6G), the Pre-B lymphoblastic leukemia cell line 697 ( Figure 6H), and against solid tumor cell lines such as the medulloblastoma cell line D283 ( Figure 7A) and DAOY ( Figure 7B),and the Glioblastoma cell line U87 ( Figure 7C) and LI373 ( Figure 7D).
  • solid tumor cell lines such as the medulloblastoma cell line D283 ( Figure 7A) and DAOY ( Figure 7B),and the Glioblastoma cell line U87 ( Figure 7C) and LI373 ( Figure 7D).
  • B7-H3.CAR vector design reported to date are second or third generation CARs: the second generation CARs preferentially include the CD28 (PMID:30753824; PMID:32728609) or 4-1 BB domain as costimulatory domain (PMID:30655315; PM ID: 30753824; PMID:32685008; PMID:32728609), whereas third generations B7-H3.CARs vector (PMID:31485480; 3381153; PMID: 32346608) preferentially include the CD28- 4.1 BB costimulatory domains.
  • CAR T-cells containing CD28.4-1 BB outperforms both third generation CAR T-cells with CD28.OX40 costimulatory domain and second generation CARs containing CD28, 0X40 and 4-1 BB domains in neuroblastoma models (see page 5 of Quintarelli and al. report (PMID: 29872565). Moreover, Andreas A. Hornbach and al. reported that CD28 outperforms compared to combined CD28-OX40 "super-stimulation" in cytokine-induced killer (CIK) cells armed with chimeric antigen receptors. (PMID: 23985696).
  • the CARs according to the present invention comprise a functional safety switch (Figure 3C), without altering the safety profile of CAR T cells ( Figure 3) or killing activity.
  • the trackable markers that are mostly used in known B7-H3.CARs are GFP, mCherry and tdTomato. These trackable markers are intracellular reporter molecules to evaluate gene transfer and expression, which can be detected in living cells without selection or staining.
  • GFP protein can induce the expansion of anti GFP T lymphocytes. This finding advices against the use of the B7-H3.CARs comprising the above mentioned trackable markers in the clinic setting (PMID: 10455440)(PMID: 27435468).
  • B7-H3.CARs constructs according to the present invention do not present this disadvantage since they comprise a ACD34 flag, represented by the extracellular domain fragment of human CD34, instead of GFP, mCherry and tdTomato.
  • constructs according to the present invention can be used to treat efficiently either B7-H3+ leukemias/lymphomas or solid B7H3+ tumour-bearing-patients.
  • autoimmune diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), multiple sclerosis (MS), Sjogren’s syndrome (SS), ankylosing spondylitis (AS).
  • SLE systemic lupus erythematosus
  • RA rheumatoid arthritis
  • MS multiple sclerosis
  • SS Sjogren’s syndrome
  • AS ankylosing spondylitis
  • the anti B7-H3 chimeric antigen receptor, the nucleotide sequence, the vector, the cell and the pharmaceutical composition according to the present invention is affective in the treatment of autoimmune diseases.
  • an anti-B7-H3 chimeric antigen receptor comprising or consisting of, from the N-terminus to the C- terminus: a) a signal peptide, b) an anti B7-H3 single chain antibody domain, c) a hinge, d) a trans membrane domain, e) a co-stimulatory signaling domain, and f) CD3Zeta chain sequence, wherein said anti B7-H3 single chain antibody domain comprises or consists of anti B7-H3 M5B14 hybridoma VL sequence and anti B7-H3 M5B14 hybridoma VH sequences linked each other by a linker, and wherein anti B7-H3 M5B14 hybridoma VL sequence comprises CDR1 sequence ENIYSY (SEQ ID NO:1 ), CDR2 sequence NAK and CDR3 sequence QHHYGTPTWT (SEQ ID NO:2), whereas anti B7-H3 M5B14 hybrid
  • anti B7-H3 M5B14 hybridoma VL sequence can comprise or consist of DIQMTQSPASLSASVGETVTITCRASENIYSYLAWYQQKQGKSPHLLVYNAKTLA EGVPSRFSGSGSGTQFSLKIKSLQPEDFGSYYCQHHYGTPTWTFGGGTKLEIK (SEQ ID NO:6)
  • anti B7-H3 M5B14 hybridoma VH sequence can comprise or consist of EVMLVESGGGLVKPGGSLKLSCAASGFTFSSYAMSWVRQTPERRLEWVATISG GGRYIYYPDNMKGRFTISRDNAKNTLYLQMSSLWSEDTAIYYCARHEDGYLDYW GQGTTLTVSS (SEQ ID NO:7).
  • Sequence SEQ ID NO:6 comprises CDR1 in position 27-32, CDR2 in position 50-52 and CDR3 in position 89-98.
  • Sequence SEQ ID NO:7 comprises CDR1 in position 26-33, CDR2 in position 51 -58 and CDR3 in position 97-106.
  • the linker which links anti B7-H3 M5B14 hybridoma VL sequence and anti B7-H3 M5B14 hybridoma VH sequence can be a short flexible linker glycines-rich with a length from 7 to 14 amino acids, such as from 7 to 12, from 7 to 10 or 8 amino acids, such as for example (G4S)2 linker GGGGSGGGG (SEQ ID NO:8), G4SG2 linker GGGGSGG (SEQ ID NO:9) or G3SG4 linker GGGSGGGG (SEQ ID NQ:10) SG4SG3 linker SGGGGSGGG (SEQ ID NO:54), (SG4)2 S linker SGGGGSGGGGS (SEQ ID NO:55), (SG4)2 SG linker SGGGGSGGGGSG (SEQ ID NO:56), (SG4)2 SG3 linker SGGGGSGGGGSGGG linker (SEQ ID NO:57), (SG4)2 SGGGGSGGGG (SEQ ID NO:
  • a linker as mentioned above is useful in order to prevent epitope masking in CAR+ cell tumor blasts, said CAR T cells being able to decrease the potential risk of tumor relapse, for example in cell B leukemia.
  • CAR T cells according to the present invention provides increased safety also in the treatment of autoimmune diseases caused by B cells producing auto-antibodies.
  • said hinge can comprise or consist of one or more of the following hinges: hinge Spacer-CD8a
  • PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFA (SEQ ID NO:11 ) (nucleotide ID NO: M12828.1 and Protein ID NO: AAB04637.1 );
  • TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD SEQ ID NO:12
  • said hinge can be linked, at the N terminus, to a trackable marker, said trackable marker being linked, optionally by a second linker, to the anti B7-H3 single chain antibody domain.
  • the trackable marker can be chosen from the group consisting of:
  • ACD34 ELPTQGTFSNVSTNVS (SEQ ID NO: 16) (nucleotide ID NO AB238231.1 and Protein ID NO: BAE46748.1 );
  • NGFR NGFR:KEACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEPCLDSVT FSDVVSATEPCKPCTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEAC RVCEAGSGLVFSCQDKQNTVCEECPDGTYSDEANHVDPCLPCTVCEDTERQLR ECTRWADAECEEIPGRWITRSTPPEGSDSTAPSTQEPEAPPEQDLIASTVAGWT TVMGSSQPWTRGTTDN (SEQ ID NO: 18) (nucleotide ID NO: AK313654.1 and Protein ID NO: BAG36408.1 ); preferably ACD34: ELPTQGTFSNVSTNVS (SEQ ID NO:16) (nucleotide ID NO AB238231.1 and Protein ID NO: BAE46748.1 ).
  • the trans membrane domain can be chosen from the group consisting of CD8aTM: CDIYIWAPLAGTCGVLLLSLVIT (SEQ ID NO:19), (nucleotide ID NO NM_001768.6 and Protein ID NO: NP_001759.3); CD28TM: FWVLVWGGVLACYSLLVTVAFIIFWV (SEQ ID NQ:20) (nucleotide ID NO: BC112085.1 and Protein ID NO: AAI12086.1 ); preferably CD8aTM: CDIYIWAPLAGTCGVLLLSLVIT (SEQ ID NO: 19), (nucleotide ID NO: NM_001768.6 and Protein ID NO: NP_001759.3).
  • the co-stimulatory signaling domain can be chosen from the group consisting of
  • CD28 cytoplasmic sequence CD28 cytoplasmic sequence
  • RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO:21 ) (nucleotide ID NO: AF222341.1 and Protein ID NO: AAF33792.1 ),
  • CD137 (4-1 BB) sequence :
  • KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL SEQ ID NO:22 (nucleotide ID NO: U03397.1 and Protein NO: AAA53133.1 ),
  • RDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI (SEQ ID NO:23) (nucleotide ID NO: NM_003327.3 and Protein NO: NP_003318.1 ), a sequence obtained by linking CD28 cytoplasmic sequence: RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO:21 ) (nucleotide ID NO: AF222341.1 and Protein ID NO: AAF33792.1 ) to
  • CD137 (4-1 BB) sequence :
  • KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO:22) (nucleotide ID NO: U03397.1 and Protein NO: AAA53133.1 ), or a sequence obtained by linking CD28 cytoplasmic sequence: RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO:21 ) (nucleotide ID NO: AF222341.1 and Protein ID NO: AAF33792.1 ), to
  • RDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI SEQ ID NO:23 (nucleotide ID NO: NM_003327.3 and Protein NO: NP_003318.1 )
  • a co-stimulatory signaling domain with CD28 cytoplasmic sequence and 0X40 sequence is preferable.
  • CD3-Zeta chain can be the following sequence RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRK NPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALH MQALPPR* (SEQ ID NO: 24) (nucleotide ID NO: J04132.1 And Protein ID: AAA60394.1 ).
  • the anti-B7-H3 chimeric antigen receptor can further comprise cytoplasmic moiety of CD8cyt, CD8a cytoplasmic (CD8a cyto): LYCNHRN (SEQ ID NO:25) (nucleotide ID NO: NM_001768.6 and Protein ID NO: NP_001759.3) between the trans membrane domain and the co-stimulatory signaling domain.
  • the signal peptide can comprise or consist of MEFGLSWLFLVAILKGVQC (SEQ ID NO:26) (nucleotide ID NO:AB776838.1 and Protein ID NO: BAN63131.1 ).
  • said anti-B7-H3 chimeric antigen receptor can comprise or consist of the following sequence:
  • AKNTLYLQMSSLWSEDTAIYYCARHEDGYLDYWGQGTTLTVSSACELPTQGTFS NVSTNVSPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAP LAGTCGVLLLSLVITLYCNHRNEFRSKRSRLLHSDYMNMTPRRPGPTRKHYQPY APPRDFAAYRSRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKIRVKFSRS ADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLY NELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
  • (M5B14)B7-H3.CAR-ACD34.CD8a.CD28.4-1 BB£ comprises: A Signal peptide MEFGLSWLFLVAILKGVQC (SEQ ID NO:26) (nucleotide ID NO:AB776838.1 and Protein ID NO: BAN63131.1 ), which is linked by the Linker (connection sequence) SR to
  • ELPTQGTFSNVSTNVS (SEQ ID NO: 16) (nucleotide ID NO AB238231.1 and Protein ID NO: BAE46748.1 );
  • PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFA (SEQ ID NO:11 ) (nucleotide ID NO: M12828.1 and Protein ID NO: AAB04637.1 );
  • CDIYIWAPLAGTCGVLLLSLVIT (SEQ ID NO: 19), (nucleotide ID NO: NM_001768.6 and Protein ID NO: NP_001759.3);
  • LYCNHRN (SEQ ID NO:25) (nucleotide ID NO: NM_001768.6 and Protein ID NO: NP_001759.3)
  • CD28 cytoplasmic sequence CD28 cytoplasmic sequence
  • RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO:21 ) (nucleotide ID NO: AF222341.1 and Protein ID NO: AAF33792.1 ),
  • MEFGLSWLFLVAILKGVQC (SEQ ID NO:26) (nucleotide ID NO:AB776838.1 and Protein ID NO: BAN63131.1 ), which is linked by the Linker (connection sequence) SR to
  • ELPTQGTFSNVSTNVS (SEQ ID NO: 16) (nucleotide ID NO AB238231.1 and Protein ID NO: BAE46748.1 );
  • PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFA (SEQ ID NO:11 ) (nucleotide ID NO: M12828.1 and Protein ID NO: AAB04637.1 );
  • CDIYIWAPLAGTCGVLLLSLVIT (SEQ ID NO: 19), (nucleotide ID NO: NM_001768.6 and Protein ID NO: NP_001759.3);
  • RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO:21 ) (nucleotide ID NO: AF222341.1 and Protein ID NO: AAF33792.1 ),
  • RDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI SEQ ID NO:23
  • CD3-Zeta chain SEQ ID NO:23
  • the present invention concerns also a nucleotide sequence comprising or consisting of a nucleotide sequence which encodes an anti-B7-H3 chimeric antigen receptor according to the above.
  • the anti B7-H3 M5B14 hybridoma VL sequence can be encoded by the nucleotide sequence GACATCCAGATGACTCAGTCGCCAGCCTCCCTATCTGCATCTGTGGGAGAAA CTGTCACCATCACATGTCGAGCAAGTGAGAATATTTACAGTTATTTAGCATGG TATCAGCAGAAACAGGGAAAATCTCCTCACCTCCTGGTCTATAATGCAAAAAC CTTAGCAGAAGGTGTGCCATCAAGGTTCAGTGGCAGTGGATCAGGCACACAG TTTTCTCTGAAGATCAAAAGCCTGCAGCCTGAAGATTTTGGGAGTTATTATTG
  • TCAACATCATTATGGTACTCCCACGTGGACGTTCGGTGGAGGCACCAAGCTG GAAATCAAA (SEQ ID NO:29), and anti B7-H3 M5B14 hybridoma VH sequence can be encoded by the nucleotide sequence GAAGTGATGCTGGTGGAGTCTGGGGGAGGTTTAGTGAAGCCTGGAGGGTCC CTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAGCTATGCCATGTC TTGGGTTCGCCAGACTCCGGAAAGGAGGCTGGAGTGGGTCGCAACCATTAG TGGTGGTGGTCGTTACATCTACTATCCAGACAATATGAAGGGGCGATTCACC ATCTCCAGAGACAATGCCAAGAACACCCTGTACCTGCAAATGAGCAGTCTGT GGTCTGAGGACACGGCCATCTATTACTGTGCAAGACATGAAGACGGCTACCT CGACTACTGGGGCCAAGGCACCACTCTCACAGTCCTCA(SEQ ID NO:30).
  • sequence SEQ ID NO:29 the sequences encoding CDR1 , CDR2 and CDR3 are the following:
  • CDR1 GAGAATATTTACAGTTAT (SEQ ID NO:31 ) in position 79-96;
  • CDR2 AATGCAAAA in position 148-156;
  • CDR3 CAACATCATTATGGTACTCCCACGTGGACG (SEQ ID NO:32) in position 265-284
  • sequence SEQ ID NQ:30 the sequences encoding CDR1 , CDR2 and CDR3 are the following:
  • CDR1 GGATTCACTTTCAGTAGCTATGCC (SEQ ID NO:33) in position 76-99;
  • CDR2 ATTAGTGGTGGTGGTCGTTACATC (SEQ ID NO:34) in position 151-174;
  • CDR3 GCAAGACATGAAGACGGCTACCTCGACTAC (SEQ ID NO:35) in position 289-294.
  • nucleotide sequence encoding anti-B7-H3 chimeric antigen receptor can be the following sequence:
  • said nucleotide sequence can further comprise a nucleotide sequence encoding a suicide gene inducible amino acid sequence linked to the nucleotide sequence encoding said chimeric antigen receptor by a nucleotide sequence encoding a 2A self-cleaving peptide.
  • the suicide gene inducible amino acid sequence can be a chimeric Caspase-9 polypeptide or comprise a herpes simplex virus thymidine kinase.
  • the polynucleotide 2A self-cleaving peptide cuts the peptide comprising the suicide gene inducible amino acid sequence and the chimeric antigen receptor in two separate peptides, i.e. the suicide gene inducible and the chimeric antigen receptor amino acid sequences.
  • the nucleotide sequence can be the following sequence
  • nucleotide sequence which encodes the sequence named also as SFG.iC9-2A-(M5B14)B7-H3.CAR-ACD34.CD8a.CD28.4-1BB£ comprises the following sequences:
  • TAAAACATCAGCTAGC (SEQ ID NQ:40) (GenBank ID MW218436.1 ), which is linked by a first linker AGAGCC to:
  • ACD34 GAACTTCCTACTCAGGGGACTTTCTCAAACGTTAGCACAAACGTAAGT (SEQ ID NO:44) (nucleotide ID NO AB238231.1 )
  • CTTTATTGCAACCATCGAAAC (SEQ ID NO:47) (nucleotide ID NO: NM_001768.6)
  • CD28 cytoplasmic sequence CD28 cytoplasmic sequence
  • nucleotide ID NO: J04132.1 The nucleotide sequence, which encodes the sequence named also as
  • SFG.iC9-2A-(M5B14)B7-H3.CAR-ACD34.CD8a.CD28.OX40? comprises the following sequences:
  • CTGAGCATGGAGCCCTGTGGCCACTGCCTCATTATCAACAATGTGAACTTCT GCCGTGAGTCCGGGCTCCGCACCCGCACTGGCTCCAACATCGACTGTGAGA AGTTGCGGCGTCGCTTCTCCTCGCTGCATTTCATGGTGGAGGTGAAGGGCG ACCTGACTGCCAAGAAAATGGTGCTGGCTTTGCTGGAGCTGGCGCAGCAGG
  • AGTGTTCACGA (SEQ ID NO:42) (nucleotide ID NO:AB776838.1 )
  • CD8a cytoplasmic CD8a cytoplasmic (CD8a cyto): CTTTATTGCAACCATCGAAAC (SEQ ID NO:47) (nucleotide ID NO: NM_001768.6)
  • CD28 cytoplasmic sequence CD28 cytoplasmic sequence
  • CD3-Zeta chain CD3-Zeta chain
  • the present invention concerns also a vector comprising the nucleotide sequence according to the above, wherein said vector is a DNA vector, a RNA vector, a plasmid, a lentivirus vector, adenoviral vector, retrovirus vector, such as y- retroviral vector, or non-viral vector.
  • the present invention concerns a cell, such as T cell, such as alfa/beta and gamma/delta T cell, NK cells, NK-T cells, macrophages or monocyte cells, comprising the anti-B7-H3 chimeric antigen receptor according to the above and/or the vector or plasmid according to the above.
  • T cell such as alfa/beta and gamma/delta T cell
  • NK cells such as alfa/beta and gamma/delta T cell
  • NK cells such as alfa/beta and gamma/delta T cell
  • NK cells such as alfa/beta and gamma/delta T cell
  • NK cells such as alfa/beta and gamma/delta T cell
  • NK cells such as alfa/beta and gamma/delta T cell
  • NK-T cells such as gamma/delta T cell
  • the cell can further comprise a suicide gene inducible amino acid sequence such as a chimeric Caspase-9 polypeptide or a herpes simplex virus thymidine kinase (HSV-TK) as a safety switch.
  • a suicide gene inducible amino acid sequence such as a chimeric Caspase-9 polypeptide or a herpes simplex virus thymidine kinase (HSV-TK) as a safety switch.
  • the chimeric Caspase-9 polypeptide can comprise or consist of: an inducible Caspase 9 (iC9):
  • YKQMPGCFNFLRKKLFFKTSAS (SEQ ID NO:52) (GenBank ID MW218436.1 and Protein ID NO: QPB74035.1 ), which is linked by a linker, such as RA, to:
  • EGRGSLLTCGDVEENPGP (SEQ ID NO:53) (nucleotide ID NO: NC_043231.1 and Protein ID NO: YP_009665206.1 ).
  • the cell can be obtained in culture conditions wherein both IL-7 and IL-15 are present, for example in the culture conditions of the activation step, transduction step and/or expansion step of the process for the preparation of said cell.
  • the present invention concerns also a pharmaceutical composition
  • a pharmaceutical composition comprising the nucleotide sequence according to the above, or the vector according to the above, or the cell according to the above together with one or more excipients and/or adjuvants.
  • the present invention concerns the anti B7-H3 chimeric antigen receptor according to the above, the nucleotide sequence according to the above, the vector according to the above, the cell according to the above, the pharmaceutical composition according to the above, for medical use.
  • the present invention concerns also the anti B7-H3 chimeric antigen receptor according to the above, the nucleotide sequence according to the above, the vector according to the above, the cell according to the above, the pharmaceutical composition according to the above, for use in the treatment of hematologic malignancies, such as for example Chronic Myeloid Leukemia (CML), Myelodysplastic syndromes (MDS), Acute Myeloid Leukemia (AML), Chronic lymphocytic leukemia (CLL), B cell Acute lymphoblastic leukemia (B-ALL), T cell Acute lymphoblastic leukemia (T-ALL), lymphomas (Non-Hodgkin's Lymphoma or Hodgkin's Lymphoma), Multiple Myeloma and solid B7H3+ tumour, such as for example Neuroblastoma, retinoblastoma, sarcoma, Ewing’s sarcoma, rhabdomyosarcoma, Osteosarcoma,
  • the present invention concerns also the B7-H3 CAR according to the above, the nucleotide sequence according to the above, the vector according to the above, the cell according to the above, the pharmaceutical composition according to the above, for use in the treatment of autoimmune diseases such as, for example, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), multiple sclerosis (MS), Sjogren’s syndrome (SS), and ankylosing spondylitis (AS).
  • SLE systemic lupus erythematosus
  • RA rheumatoid arthritis
  • MS multiple sclerosis
  • SS Sjogren’s syndrome
  • AS ankylosing spondylitis
  • the anti B7-H3 chimeric antigen receptor, nucleotide sequence, vector, cell, pharmaceutical composition according to the present invention can be advantageously administered by systemic administration, also in the treatment of brain tumors.
  • Figure 1 shows four B7-H3.CAR SFG clinical grade “third” generation of retrovirus vector.
  • A-B The scFv of B7H3 (Ne97 mAb) was cloned in frame with CD8aTM, CD28 cytoplasmic moiety, and a second costimulatory domain represented by either 4-1 BB (A) or 0X40 (B), as well as the signaling domain CD3- zeta chain (Q.
  • ACD34 was added.
  • C-D The scFv of B7H3 (M5B14) was cloned in frame with CD8aTM, CD28 cytoplasmic moiety, and a second costimulatory domain represented by either 4-1 BB (C) or 0X40 (D), as well as the signaling domain CD3-zeta chain ( .
  • ACD34 was added as a trackable marker.
  • FIG. 2 shows that B7-H3.CAR T cells with CD28.OX40 or CD28.4-1 BB costimulation exhibit high transduction level.
  • A Flow-cytometry analyses in a representative donor showing chimeric antigen receptor (CAR) expression by detection of membrane ACD34 in non-transduced (NT) T cells (negative control; left panel), and T cells genetically modified with NE97. B7-H3. CAR-28.4-1 BB , NE97.B7-H3. CAR-28. OX40 , M5B14.B7-H3. CAR-28.4-1 BB and M5B14.B7- H3. CAR-28.0X40 ⁇ . growth in IL-2 (from the second to the fifth square panel, respectively).
  • MFI Median Fluorescence Intensity
  • Figure 3 shows that B7-H3.CAR T cells with CD28.OX40 or CD28.4-1BB costimulation exhibit similar in vitro proliferation and safety profile upon cytokine stimulation.
  • A Fold expansion of NT T-cells (black dots on dashed line), NE97.B7-H3. CAR-28.4-1 BBC T-cells (empty dots), NE97. B7-H3. CAR-28. OX40C T- cells (empty triangles), M5B14.B7-H3. CAR-28.4-1 BBC T-cells (black dots) and M5B14.B7-H3.
  • CAR-28. OX40C T-cells black triangle
  • (D) The Expression of Vector Copy Number of B7-H3.CAR T- cells, at day +15 was below 12, (range 1 ,1 -11 ,5). Data from six healthy donors (HDs) are expressed as average ⁇ SD. ****p-value ⁇ 0,0001 .
  • FIG. 4 shows B7-H3 (CD276) Expression in solid and hematological tumors cell lines.
  • the expression of B7-H3 antigen was evaluated, by flow cytometry.
  • B7-H3 is expressed in (A) two out of four lymphoma cell lines; (B) one in four B-acute lymphoblastic leukemia cell lines; (C) four out of five Acute Myeloid Leukaemia; (D) one chronic myeloid leukaemia; (E) all evaluated neuroblastoma cell lines; (F) all evaluated sarcoma cell lines (specifically Ewing’s sarcoma, Embryonal rhabdomyosarcoma (ERMS), alveolar Rhabdomyosarcoma (ARMS) and Osteosarcoma cell lines) ; (G) all evaluated Brain tumours (specifically Glioblastoma and Medulloblastoma cell lines); (H) all evaluated pancreatic cancer cell lines; (I) all evaluated Col
  • Figure 5 shows that both scFv in B7-H3.CAR T cells (NE97 and M5B14), expressing either CD28.4-1BB or CD28.OX40 costimulatory domains, show comparable short-term cytotoxic effect in vitro experiment.
  • In vitro 51 Cr release assay showing that NE97.B7-H3.
  • CAR-28.4-1 BB T-cells solid line with empty dots
  • NE97.B7-H3.CAR-28.OX40C T-cells solid line with empty triangles
  • CAR-28.4-1 BBC T-cells (solid line with black dots) and M5B14.B7- H3.
  • OX40C T-cells (solid line with black triangle) exert cytolytic activity against B7-H3+ Hodgkin Lymphomas HDLM-2 (A), but not NT T cells (dotted line with black square), B7-H3 negative Hodgkin Lymphomas L428 (B). They also kill with the same efficiency B7-H3+ Acute Myeloid Leukemia OCI-AML3 (C). Note that, at a very low E:T ratio (5:1 ), NE97.B7-H3.CAR-28.OX40C CAR T-cells kill the SHSY5Y NB cell line with significant higher efficiency respect to M5B14. B7- H3. CAR-28.4-1 BB or M5B14.B7-H3.CAR-28.OX40C T cells (D). Data from six healthy donors (HDs) are expressed as average ⁇ SD. * p-value ⁇ 0.05.
  • Figure 6 shows that long-term co-culture of both NE97 and M5B14 scFv B7-H3.CAR T cells, expressing either CD28.4-1BB or CD28.OX40 costimulatory domains, confirm their specific cytotoxic potency against different extracranial B7-H3+ tumor cell lines. Average representation of remaining tumor cells, after 6 days-coculture at the ratio E/T 1 :1 with NT T-cells, NE97.B7-H3. CAR-28.4-1 BB T-cells, NE97.B7-H3.CAR-28.OX40C T-cells, M5B14.B7-H3. CAR-28.4-1 BBC T-cells and M5B14. B7-H3.
  • CAR-28 OX40C T-cells, growth in IL2.
  • A-B Neuroblastoma cell lines;
  • C Ewing sarcoma A673 cell line;
  • D ERMS RD cell line;
  • E Hodgkin Lymphoma HDML-2 cell line;
  • F Acute Myeloid Leukemia OCI-AML3;
  • G Acute monoblastic/monocytic leukemia;
  • H Pre-B lymphoblastic leukemia 697.
  • Data from six healthy donors (HDs) are expressed as average ⁇ SD.
  • Figure 7 shows that long-term co-culture of both NE97 and M5B14 scFv in B7-H3.
  • CAR T cells expressing either CD28.4-1BB or CD28.OX40 costimulatory domains, confirm their potent specific cytotoxic potency against B7-H3+ brain tumor cell lines. Average representation of remaining tumor cells, after 6 days-coculture at the ratio E/T 1 :1 with NT T-cells, NE97.B7-H3. CAR- 28.4-1 BB T-cells, NE97.B7-H3. CAR-28.
  • OX40 T-cells M5B14.B7-H3.CAR-28.4- 1 BB T-cells and M5B14.B7-H3.CAR-28.OX40C T-cells, growth in IL-2.
  • A-B Medulloblastoma cell lines D283 and DAOY;
  • C-D glioblastoma cell lines U87 and U373.
  • Data from six healthy donors (HDs) are expressed as average ⁇ SD. * p- value ⁇ 0.05; ** p-value ⁇ 0.01 ; *** p-value ⁇ 0.001 and **** ⁇ 0.0001.
  • Figure 8 shows that IL-7/IL-15 used in culture conditions did not influence the kinetic expansion of NE97. B7-H3.CAR T cells.
  • Figure 9 shows that cytokines used in culture conditions significantly improve in vitro killing activity of NE97.B7-H3.CAR T-cells expressing either CD28.4-1BB or CD28.OX40 costimulatory domains.
  • Average representation of remaining tumor cells of several tumor cell lines as the MB cell line DAOY (A); the neuroblastoma cell line SHSY5Y (B); the Osteosarcoma (OS) cell lines (C-E): 143B (C), HOS (D) and O2-OS (E); the ARMS cell lines (F-G): RH30 (F) and RH41 (G); the ERMS RD (H), the Ewing’s sarcoma A673 (I) and SK-ES-1 (J) and the Non- Hodjkin Lymphoma Karpas 299 (K), after 6 days co-culture, at the ratio E/T 1 :1 , with NT T-cells, NE97.B7-H3.
  • CAR-28.4-1 BBC T-cells, NE97.B7-H3.CAR-28.OX40C T- cells growth in IL2 (white bars) (A) or IL7/IL15 (B-K) (black bars). Data from six healthy donors (HDs) are expressed as average ⁇ SD. * p-value ⁇ 0.05; ** p- value ⁇ 0.01 ; *** p-value ⁇ 0.001 and **** ⁇ 0.0001 .
  • Figure 10 shows that both (IL7/IL15) NE97.B7-H3.CAR T-cells, expressing either CD28.4-1BB or CD28.OX40 costimulatory domains, kill very efficiently also adult solid tumor cell lines.
  • Data from four healthy donors (HDs) are expressed as average ⁇ SD. * p-value ⁇ 0.05; ** p-value ⁇ 0.01.
  • Figure 11 shows in vitro long-term co-culture potency assay to evaluate functional activities of NE97.B7-H3.CAR T-cells.
  • the B7-H3+ MB DAOY tumor cell line was used as target.
  • Data from 4 HDs are expressed as average ⁇ SD for (D); * p-value ⁇ 0.05; ** p-value ⁇ 0.01.
  • Figure 12 shows that NE97.B7-H3.CAR-28.OX40 CAR T-cells produce higher CAR-T-derived cytokines respect to NE97.B7-H3.CAR-28.4-1BBC T- cells when co-cultured with B7-H3+ tumor cell lines RH30 (A-D) or A673 (E-H).
  • a and E GranB
  • B and F IFN-y
  • C and H TNF-a
  • D and G IL-2 production were analysed in supernatants collected 24-hours after tumor addition to the culture.
  • Data from six healthy donors (HDs) are expressed as average ⁇ SD. * p-value ⁇ 0.05; ** p-value ⁇ 0.01 ; ***p-value ⁇ 0.001 and **** ⁇ 0.0001.
  • Figure 13 shows that NE97.B7-H3.CAR T-cells proliferate specifically when co-cultured with B7-H3+ tumor cell line or upon activation by B7-H3 Ligand.
  • A Proliferation assay based on 3H-thymidine incorporation of NT T-cells or NE97.B7-H3.CAR T-cells cells, stimulated for five days, with irradiated RH30 tumor cells (45 Gy), or the cytokine cocktail (IL7/IL15), or the B7-H3 ligand.
  • Data represent results from 3 HDs.
  • Figure 14 shows the results obtained with sarcoma mouse model to evaluate anti-tumor activity of NE97.B7-H3.CAR T-cells generated and expanded in the presence of IL7/IL15.
  • A) The cartoon shows the in vivo xenograft immunodeficient mouse model, in which the ARMS cell lines RH30-GFP-FF-Luc cells were systemically infused in NSG mice. Effector cells were infused i.v. at the time of tumor establishment (Day 3), as assessed by IVIS Imaging.
  • B Exemplificative IVIS Imaging of tumor growth from day +2 to end-of-experiment for mice treated with NT T-cells or NE97.B7-H3.CAR T-cells.
  • C Average of tumor bioluminescence of xenograft mice treated with NT T-cells (dotted line), with NE97. B7-H3.
  • CAR-28.4-1 BB T-cells (dotted line with black dots) or with NE97.B7- H3.
  • CAR-28. OX40 T-cells (solid line with black dots).
  • OS Kaplan-Meier survival curve
  • Figure 15 shows the results obtained with MB intracranial mouse model to evaluate the antitumor activity of NE97.B7-H3.CAR T-cells.
  • A) The cartoon shows NSG mice engrafted with MB cell line D283.GFP-FF-luciferase in the brain, by stereotaxic system. Effector cells were infused i.v. at the time of tumor establishment (Day 14), as assessed by IVIS Imaging. Mice underwent periodical blood collections.
  • B Exemplificative IVIS Imaging of tumor growth from day +14 to end-of-experiment for mice treated with NT T-cells or NE97.B7-H3.CAR T-cells.
  • CD3+T-cells in mice treated with NT T-cells (dotted line with black dots), NE97.B7- H3.
  • CAR- 28. OX40C T-cells (solid line with black triangle).
  • CAR-28.4-1 BBC T-cells (white with black polka dots violin plot), and NE97.B7-
  • a clinical grade “third” generation of retrovirus bicistronic vector SFG have been designed, allowing the simultaneous expression of two transgenes, namely iC9 suicide gene and the cassette anti-B7-H3 single-chain variable fragment (scFv), derived from a murine antibody of IgG (Ne97) class or IgM (M5B14), linked via a codon optimized human CD8 spacer-transmembrane domain, to the codon optimized signaling costimulatory domain CD28, the codon optimized signaling costimulatory domain 4-1 BB (CD137) or 0X40 and CD3- .
  • scFv single-chain variable fragment
  • the iC9 gene contains the intracellular portion of the human caspase 9 protein, a pro-apoptotic molecule, fused to a drug-binding domain derived from human FK506-binding protein FKBP12.
  • a T2A self-cleaving peptides sequence separate iC9 from CAR sequence.
  • the scFv Ne97 or M5B14 is cloned in frame with codon optimized CD34 derived epitope of 16 aa (as trackable marker), linked by spacer of 40 aa to bind the codon optimized human CD8-transmembrane domain (CD8aTM) of 23 aa.
  • the signal run from extracellular portion of B7-H3 scFv to intracellular portion of CD3- chain (113aa) through two costimulatory molecules: CD28 endodomain (41 aa) and 4-1 BB endodomain (42aa) or 0X40 endodomain (36 aa).
  • Retroviral supernatant was generated in 293T-cells (PMID:32381575, PMID:20686963) and quantified by Retro-XTM qRT-PCR Titration Kit (Takara) to be used at 10 9 retrovirus-copies/0.5x106 T-cells.
  • the supernatant was used to transduce primary T cells derived from peripheral blood mononuclear cells of healthy donors (Ethical Committee Approval N°969/2015 prot. N°669LB).
  • T lymphocytes were activated with immobilized OKT3 (1 pg/ml, e-Bioscience lnc.;San Diego, CA, USA) and anti-CD28 (1 pg/ml, BD Biosciences, Europe) antibodies in the presence of interleukin-2 (IL-2) or combination of recombinant human interleukin-7 (IL-7, 10 ng/ml; R&D; USA) and recombinant human interleukin-15 (IL-15, 5 ng/ml; R&D).
  • IL-2 interleukin-2
  • IL-7 recombinant human interleukin-7
  • IL-15 recombinant human interleukin-15
  • Activated T cells were transduced on day 3 in 24-well plates pre-coated with recombinant human RetroNectin (Takara- Bio.
  • the T cells are expanded in “CTL complete medium” containing 45% RPMI 1640 and 45% Click’s medium (Sigma- Aldrich, Co.; Usa) supplemented with 10% FBS and 2 mM Glutamax, and fed twice a week with the specific above described cytokines (PMID: 29872565).
  • CTL complete medium containing 45% RPMI 1640 and 45% Click’s medium (Sigma- Aldrich, Co.; Usa) supplemented with 10% FBS and 2 mM Glutamax, and fed twice a week with the specific above described cytokines (PMID: 29872565).
  • eGFP-FFLuc The retroviral vector encoding eGFP-Firefly-Luciferase (eGFP-FFLuc) was used in selected experiments to label B7-H3 positive (B7-H3+) or B7-H3 negative (B7-H3-) tumor cells:
  • Ewing sarcoma cell lines A673 and SK-ES-1
  • Embryonal Rhabdomyosarcoma (ERMS) cell line RD Embryonal Rhabdomyosarcoma (ERMS) cell line RD
  • Osteosarcoma (OS) cell lines 143B; HOS; LI2-0S Pancreatic carcinoma cell line (Mia PaCa-2) Colon carcinoma cell line (HCT-116) B7-H3- (negative) tumor cell lines: Hodgkin’s Lymphoma cell line L428
  • Hodgkin's Lymphomas (HL) HDML-2 and L428 and the B cell precursor leukemia Ph+ BV173, the TOM-1 were obtained from DSMZ.
  • the B cell precursor leukemia 697 and RS4;11 were obtained from DSMZ.
  • Burkitt Lymphoma Daudi was obtained from ATCC.
  • Non-Hodgkin’s Lymphoma (NHL) Karpas 299 was obtained from Sigma-Aldrich.
  • the Acute Myeloid Leukemia OCI-AML3, MOLM-13 were obtained from DSMZ.
  • the Acute Myeloid Leukemia MV-4;11 , HL-60 and THP-1 were obtained from DSMZ.
  • the myelogenous leukemia cell line K562 was from LGC Standards-ATCC.
  • the neuroblastoma cell lines SHSY5Y, IMR-32, SK-N-BE(2), SK- N-SH were obtained from LGC Standards-ATCC.
  • the GD2-negative subclone of SHSY5Y [SHSY5Y GD2(neg)] cell line has been selected with the BD FACSAria III sorter (PMID: 29872565).
  • the neuroblastoma cell lines LAN-1 was obtained from DSMZ.
  • the Ewing’s sarcoma cell lines SK-ES-1 and A-673 were obtained from LGC Standards-ATCC.
  • the ERMS RD was obtained from LGC Standards-ATCC.
  • the ARMS RH30 and RH41 were obtained from DSMZ.
  • the OS cell lines: 143B, HOS, U-2OS and SAOS-2 were obtained from LGC Standards-ATCC.
  • the Glioblastoma U87 was obtained from LGC Standards-ATCC.
  • the medulloblastomas DAOY and D283 were obtained from LGC Standards-ATCC.
  • the embryonic kidney 293T cell line were obtained from LGC Standards-ATCC.
  • the pancreatic tumor cells PANC- 1 , MIAPaCa-2, BxPC-3, CFPAC-1 and AsPC1 were obtained from LGC Standards- ATCC.
  • the colon cancer cells HT-29, HCT-116, CaCo-2, SW480, DLD-1 , and Lovo were obtained from LGC Standards-ATCC.
  • the breast cancer SK-BR-3 was obtained from LGC Standards-ATCC. Cells were maintained in a humidified atmosphere containing 5% CO2 at 37°C. All cell lines were routinely tested for mycoplasma and for surface expression of target antigens. All cell lines have been authenticated by STR analysis in the certificated lab "BMR Genomics s.r.l.”
  • T-cell receptor (TCR)-Vp repertoire on NT T cells and CAR-T cells was evaluated at day+15 and day+30, using a panel of 24 different TCR V
  • NT and B7-H3.CAR T lymphocytes were plated at 0.2x10 6 cells/well in 24-well plates at the indicated E:T ratios. Following 6 days of incubation at 37 °C, tumor cells and T cells were collected and residual tumor cells and T cells assessed by fluorescence-activated cell-sorting (FACS) analysis based on CD3 expression (Effector T cells) and GFP (tumor cell line).
  • FACS fluorescence-activated cell-sorting
  • Total DNA was purified by QIAamp DNA Mini Kit (Qiagen) according to the manufacturer’s instructions.
  • TaqMan primer/probes were designed specific for the inducible Caspase 9 (iC9) suicide gene.
  • qPCR was performed by using the 7900 HT fast-Real Time-PCR System and ViiA7 system (ThermoFisher Scientific, USA) and TaqMan Gene Expression Master Mix (ThermoFisher Scientific, USA).
  • T cells and B7-H3.CAR T-cells were exposed to 10 nM AP1903 (cat#6130, Bio-techne brand) for 48 hours and residual viable cells were stained with Annexin- V/7AAD (BD Pharmingen) and analysed by flow cytometry.
  • mice engrafted with 0.2x10 6 of MB cell line D283.GFP-FF-luciferase in the brain, by stereotaxic system. After tumor engraftment, mice received only one i.v. injection of effector T cells (10x10 6 /mouse). This means that no more than one infusion of the effector cells or fractionated dosages are necessary.
  • Tumor growth was evaluated using IVIS imaging system (PerkinElmer, USA), Briefly, a constant region of interest was drawn over the mouse and the intensity of the signal measured, every week, as total photon/sec/cm2/sr (p/s/cm2/sr), as previously described (PMID: 20686963). Mice were maintained in the animal facility at Plaisant Castel Romano (Rome, Italy). All in vivo experiments were in compliance with the ethical international, Ell and national requirements and were approved by the Italian Health Ministry (N°88/2016-PR). The circulating human T cells were evaluated periodically in mice peripheral blood.
  • the transduction efficiency of primary T cells, growth in IL-2 was similar between the two types of third generation ( Hl ) B7-H3.CARs constructs (namely NE97. B7-H3. CAR-28.4-1 BB and NE97.B7-H3.CAR-28.OX40C or M5B14.B7- H3. CAR-28.4-1 BB and M5B14. B7-H3. CAR-28.0X400 ( Figure 2A and Figure 2B). However, NE97.B7-H3. CAR-28.
  • NE97.B7-H3.CAR-28.OX40C CAR T-cells showed more CAR molecules surface expression, as indicated by significant higher Median Fluorescence Intensity (MFI) (20702, 25 ⁇ 11047,47) respect to the other three CARs: namely NE97.B7-H3.
  • MFI Median Fluorescence Intensity
  • CAR-28.4-1 BBC (12080,75 ⁇ 9306,41 ), M5B14.B7-H3.CAR-28.4-1 BBC (3751 ,00 ⁇ 1856,51 ) and M5B14.B7-H3.
  • CAR-28. OX40C CAR T- cells present more CAR molecules on the surface (membrane) of the effector cell in comparison to the other CAR T cells and therefore NE97.B7-H3. CAR-28.
  • OX40C CAR T-cells are better armed to recognize and kill cancer cells.
  • B7-H3.CAR molecules did not induce any significant proliferative change in genetically modified T cells as compared to non-transduced (NT) control T cells and was superimposable during the first two weeks of culture (Figure 3A), and did not induce any TCR V
  • B7-H3 (CD276, B7H3, B7RP-2) antigen was evaluated, by flow cytometry, on several haematological tumor cell lines. Specifically, B7-H3 is expressed in two out of 4 lymphoma lines, (both Hodgkin lymphomas (HL) and nonHodgkin lymphomas (NHL) ( Figure 4A), in one out of 4 B-acute lymphoblastic leukemia cell line ( Figure 4B), in four out of five acute myeloid leukemia cell lines (Figure 4C) and in one Chronic myeloid leukaemia (CML) ( Figure 4D).
  • HL Hodgkin lymphomas
  • NHL nonHodgkin lymphomas
  • Figure 4C B-acute lymphoblastic leukemia cell line
  • Figure 4C chronic myeloid leukaemia
  • Figure 5 shows that both scFv HI B7- H3.CAR T cells (NE97 and M5B14), expressing either CD28.4-1 BB or CD28.OX40, selectively kill with the same efficiency B7-H3+ Hodgkin Lymphomas HDLM-2 ( Figure 5A), but not the B7-H3 negative Hodgkin Lymphomas L428 ( Figure 5B). They also kill with the same efficiency B7-H3+ Acute Myeloid Leukemia OCI-AML3 ( Figure 5C).
  • This assay evaluates the ability of the effector cell to recognize and kill cancer cells after a few hours of contact (6 hours). Although they are all equally active in the ratios 40:1 , 20:1 and 10:1 ; however, when NE97.B7-H3. CAR-28. OX40C CAR T-cells were co-cultured at the lower E:T ratio (5:1 ), kill higher percentage of cancer cells (42.5%), therefore they are more efficient than the others constructs.
  • IH NE97.B7- H3.CAR T cells expressing either CD28.4-1 BB or CD28.OX40 significantly control, with higher efficiency, the tumor growth of NB tumor cell line SHSY5Y ( Figure 6A) and IMR-32 ( Figure 6B).
  • B7-H3.CARs have the suicide gene, interesting to note that, all four HI B7-H3.CAR T cells kill with the same efficiency several lymphoma and leukaemia cell lines as: the Hodgkin Lymphomas cell lines: HDML-2 ( Figure 6E), the acute myeloid leukemia (AML FAB M4) cell line OCI-AML3 ( Figure 6F), the acute monoblastic/monocytic leukemia cell line MV4-11 ( Figure 6G) and the B cell precursor leukemia cell line 697 ( Figure 6H).
  • HDML-2 the acute myeloid leukemia (AML FAB M4) cell line OCI-AML3
  • Figure 6G the acute monoblastic/monocytic leukemia cell line MV4-11
  • B cell precursor leukemia cell line 697 Figure 6H.
  • Table 7 summarizes the long-term co-culture results (as % of residual tumor cells) collected for all in B7-H3.CAR T cells co-cultures with solid tumours as neuroblastoma (NB) and sarcoma cell lines. T test value are also reported.
  • Table 8 summarizes the long-term co-culture results (as % of residual tumor cells) collected for all NI B7-H3.CAR T cells co-cultures with brain tumours as Glioblastoma and medulloblastoma cell lines. T test value are also reported.
  • Table 9 summarizes the long-term co-culture results (as % of residual tumor cells) collected for all B7-H3.CAR T cells co-cultures with lymphoma tumor cell, acute myeloid leukaemia, acute monoblastic/monocytic leukaemia and lymphoblastic leukaemia cell lines. T test values are also reported.
  • GRANB serine protease Granzyme B
  • both NT T-cells and CAR T-cells proliferate in the presence of IL-7/IL-15 cytokines; but only HI NE97.B7-H3.CAR T-cells significantly uptake the 3H-thymidine when coculture with irradiated RH30 cell lines or the B7-H3 ligand.
  • both HI NE97.B7-H3.CAR T-cells were selected for the next step, e.g the in vivo experimental evaluation in xenograft mouse model.
  • ARMS-tumor-bearing mice treated with IH NE97.B7-H3.CAR T-cells independently from costimulatory domains, showed average survival longer (undefined days for mice treated with either NE97.B7-H3. CAR-28.4-1 BB or NE97.B7-H3. CAR-28. OX40C T-cells) compared with mice treated with NT T-cells ( Figure 14D, median survival equal to 49 days).
  • mice were engrafted with D283.GFP-FF-luciferase tumor cells in the brain, by stereotaxic system. After tumor engraftment, mice were treated i.v. with effector NT or HI NE97.B7-H3.CAR T-cells (Figure 15A).
  • CAR T-cells showed a superior anti-tumor activity compared to NT T-cells, which resulted in a significant and rapid reduction of tumor bioluminescence after 30 days from the treatment: 2.8e9 ⁇ 4.7e8 p/sec/cm2/sr for mice treated with NT T-cells vs 6.5e8 ⁇ 6.9e8 p/sec/cm2/sr for mice treated with NE97.B7-H3.
  • B7-H3.CAR T-cells in particular M5B14.B7-H3.CARs, are very active against B7-H3+ leukemias/lymphomas and solid tumor cell lines.

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Abstract

La présente invention concerne un vecteur comprenant la cassette codant pour le gène CAR B7-H3 obtenu à l'aide des fragments variables de chaîne unique (scFv) de l'anticorps IgM monoclonal M5B14, un procédé de production de celui-ci et des cellules effectrices génétiquement modifiées de CAR B7-H3 (telles que des lymphocytes T ou des cellules innées telles que des cellules NK et NK-T) pour le traitement de tumeurs B7-H3-positives (CD276) telles que des tumeurs malignes lymphoïdes, de la leucémie, des tumeurs solides extra-crâniennes et du SNC, et des maladies auto-immunes.
PCT/IT2023/095002 2022-12-14 2023-12-13 Cellules effectrices de récepteur antigénique chimérique (car b7-h3) spécifiques de b7-h3 dérivées d'igm pour le traitement de tumeurs cd276+ et de maladies auto-immunes WO2024127444A1 (fr)

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