EP4658298A1 - Lymphocytes exprimant des car anti-cd86 pour thérapie tumorale ciblée - Google Patents

Lymphocytes exprimant des car anti-cd86 pour thérapie tumorale ciblée

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Publication number
EP4658298A1
EP4658298A1 EP24702779.0A EP24702779A EP4658298A1 EP 4658298 A1 EP4658298 A1 EP 4658298A1 EP 24702779 A EP24702779 A EP 24702779A EP 4658298 A1 EP4658298 A1 EP 4658298A1
Authority
EP
European Patent Office
Prior art keywords
car
cells
cell
lymphocyte
domain
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP24702779.0A
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German (de)
English (en)
Inventor
Sebastian KOBOLD
Adrian GOTTSCHLICH
Ruth GRÜNMEIER
Stefan Endres
Carsten MARR
Moritz Thomas
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Ludwig Maximilians Universitaet Muenchen In Vertretung Des Freistaates Bayern
Helmholtz Zentrum Muenchen Deutsches Forschungszentrum fuer Gesundheit und Umwelt GmbH
Original Assignee
Ludwig Maximilians Universitaet Muenchen In Vertretung Des Freistaates Bayern
Helmholtz Zentrum Muenchen Deutsches Forschungszentrum fuer Gesundheit und Umwelt GmbH
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Publication of EP4658298A1 publication Critical patent/EP4658298A1/fr
Pending legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/4224—Molecules with a "CD" designation not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], 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 [IG], 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
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
    • A61K2239/48—Blood cells, e.g. leukemia or lymphoma
    • 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)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C07K2319/41—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a Myc-tag

Definitions

  • the present invention relates to the recognition of CD86 as a marker of hematological cancer and thus relates to CD86 targeting agents for the treatment of such cancers, in particular, acute myeloid leukemia (AML), Hodgkin's lymphoma (HL) and non-Hodgkin's lymphoma (NHL).
  • the invention in particular encompasses a lymphocyte recombinantly expressing a chimeric antigen T cell receptor (CAR) comprising an antigen binding domain that specifically binds CD86 for use in the treatment of such cancers, as well as also encompassing the CAR construct, i.e., comprising an antigen binding domain that specifically binds CD86.
  • CAR chimeric antigen T cell receptor
  • T cells have been established as major target structures and effectors in oncology (Kobold et al., 2015). Over the last few years, immunotherapeutic strategies have emerged as powerful treatment option for patients suffering from chemotherapy-refractive, relapsed diseases, especially in hematological malignancies (Waldman, Fritz & Lenardo, Nat Rev Immunol (2020);20:651-668; Majzner & Mackall, Nat Med (2019);25:1341-1355). Hematological malignancies are cancers that affect the blood, bone marrow, and lymph nodes. This classification includes various types of leukemia such as acute myeloid leukemia (AML), lymphoma (Hodgkin's lymphoma (HL) and non-Hodgkin's lymphoma (NHL)).
  • AML acute myeloid leukemia
  • HL lymphoma
  • NHL non-Hodgkin's lymphoma
  • immune checkpoint blockade In the complex field of immunotherapy, immune checkpoint blockade (ICB) can be considered the most advanced. It has been demonstrated that T cells can be activated and targeted against a plethora of malignant diseases, however, the efficiency of these checkpoint inhibitors in treatment of hematological cancer remains elusive (Boekstegers et al., Bone Marrow Transplant. (2017); 52(8):1221-1224).
  • T cells may be used successfully as direct therapeutic targets.
  • TCR tumor-associated antigen-specific T cell receptor
  • CAR chimeric antigen receptor
  • Such "CAR T cells” are considered to couple the specificity of an antibody with the destructive force of T cell effector functions (Benmebarek et al., Int J Mol Sci. (2019); 14; 20(6)), thereby constituting a powerful approach to adoptive cell therapy (ACT).
  • ACT adoptive cell therapy
  • CD33 is a surface marker broadly expressed on myeloid cells and initial studies using anti-CD33-CAR-T cells have shown promising anti-tumor potency. However, it is also associated with severe side-effects, similar to those of anti-CD19-CAR-T cells (Wang et al., Mol Ther. (2015); 23(1):184-91). The side-effects are believed to be caused by an insufficient target specificity.
  • An ideal target structure for AML should be expressed on AML cells as broadly and homogenously as possible, but not on cells of the healthy hematopoiesis (or at least only on infrequently occurring subtypes).
  • a similar teaching is applicable to CD30-based CAR T cell treatment which is described as at risk for on-target off-tumor toxicity (Brudno J.N. et al., Blood (2022),140(Supplement 1):12731-12732; Hornbach A.A. et al., Mol Ther. (2016); 24(8):1423- 1434).
  • target structures for hematological cancers are also expressed on cells of the healthy hematopoiesis or related cell types (which also explains the majority of the expected and observed toxicity associated with the targeting of such antigens by the various therapies tested thus far).
  • target structures that are not significantly expressed on healthy cells have the disadvantage that they are not typically uniformly expressed on hematological cancer cells, or are only expressed in specific cancer subtypes limiting general applicability.
  • the expected benefit of therapies targeting the more restricted markers is reduced, and a long-lasting therapeutic effect is prevented. Accordingly, what is needed is the identification of a more promising target molecule with limited risk for on-target off-tumor toxicity and antigen escape mechanisms.
  • CD86 was identified as a broadly expressed targetstructure of hematological cancers.
  • CD86 was known to be expressed on subpopulations of hematologic cancer.
  • CD86 was recognized primarily for its role in the CD80-CD86-CTLA-4-CD28 axis and, thus, investigated for its immunomodulatory functions (Van Gool et al., Leukemia (1997);11:846-851)).
  • Therapeutic efforts regarding the CD80-CD86-CTLA-4-CD28 axis have included examining treatment efficacy of CTLA-4-T cells, however, these cells were described to potentially present a risk for clinical development even in the autologous setting (Lin et al, Front. Immunol.
  • CD86 was thought to be mainly expressed on anti-tumorigenic Mi macrophages, while being only lowly expressed on M2 macrophages (Bertani FR et al., Scientific Reports (2017);7:8965, Jablonski KA et al., PLoS One (2015);10(12):e0145342).
  • Direct targeting of CD86 using for example CAR T cells or bispecific antibodies has not yet been described (Kennedy et al., Nat Immunol. (2023); 23: 1365-1378).
  • CD86 is not recognized in the prior art as a suitable target structure for the treatment of hematological cancers (MacKay et al., Nat Biotechnol (2020); 38, 233-244).
  • the present inventors have unexpectedly found that CD86 provides a surprisingly effective target for T-cell-based therapies - even in refractory cancers where known therapies have failed.
  • CD86 is shown to be expressed on three types of hematological cancers, i.e., on various subsets of AML, HL and NHL cells, despite minimal expression ( ⁇ 2% of cells) on pre-defined critical cell clusters. Furthermore, the inventors have demonstrated that CD86 is highly expressed across polarized tumor-infiltrating macrophage subtypes, contrary to the reports in the art which described CD86 to be mainly expressed on anti- tumorigenic Ml macrophages. Targeting such CD86-expressing immuno-suppressive cell populations that contribute to tumor progression can improve anti-cancer activity of the invention, e.g. reducing the immunosuppression on adoptively transferred CAR T cells.
  • CD86 as a ubiquitous target structure in AML, HL and NHL with limited expression on normal cells, including the provision of efficient anti-CD86-CAR constructs, and anti-CD86-CAR lymphocytes with demonstrated in vitro and in vivo efficacy as well as the therapeutic use thereof.
  • the present invention provides a lymphocyte recombinantly expressing a chimeric antigen T cell receptor (CAR) for use in the treatment of cancer characterized by the expression of Cluster of Differentiation 86 (CD86, see, e.g. UniProt accession no: P42081 and Gene Bank gene ID: 942).
  • CD86 Cluster of Differentiation 86
  • the CAR construct comprises an extracellular domain that specifically binds CD86, a transmembrane domain, and an intracellularT cell activating domain.
  • the term recombinantly expresses the CAR is used as commonly understood in the art, indicating that the cell and/or its progenitor cell/cell line has been genetically engineered to express the CAR construct.
  • the CAR T cell disclosed herein comprises nucleic acid sequences not endogenously found in T cells, e.g., comprising promoter sequences operably linked to cDNA sequences encoding one or more portions of the CAR as disclosed herein such as (i) an extracellular domain that specifically binds CD86, (ii) a transmembrane domain and (iii) an intracellular T cell activating domain.
  • the extracellular domain of the CAR as described herein comprises an antigen binding region specific for CD86.
  • the antigen binding region as described herein may be any moiety providing specificity for the antigen CD86 or any epitope thereof but is preferably an antigen-binding region derived from an antibody including but not limited to antigen binding fragments derived from the Fv domain.
  • Exemplary antigen-binding regions derived from an antibody Fv domain include (but are not limited to) paired heavy and light chain variable domains, such as Fab, Fab', F(a b')z, and Fv fragments as well as recombinant constructs such as single-chain Fv domains, known in the art as scFvs.
  • the antibody-derived antigen-binding region is an scFv.
  • Any scFv known in the art or described herein specific for CD86 can be used in the construction of the CAR or lymphocyte and/or in the treatment of cancer as disclosed herein.
  • CD86-specific murine scFvs are known in the art and include that derived from clone 3D1 as disclosed in US2002/0176855 (SEQ ID NO:1), which may be encoded, for example, by SEQ ID NO:2, and/or that derived from a cell expressing SEQ ID NO:2.
  • a further non-limiting example of a CD86-specific murine scFv that may be used in the context of the invention has SEQ ID NO:24 (referenced herein as "2D5"), which may be encoded, for example, by SEQ ID NO:25, and/or that derived from a cell expressing SEQ ID NO:25.
  • the scFv may be a murine, human or humanized scFv. Humanized versions of known murine scFv sequences can readily be generated using routine procedures in the art.
  • Antigen-binding regions derived from an antibody as used herein also include antibody antigen binding fragments comprising a single, unpaired heavy or light chain variable domain as known in the art that retains the ability to specifically and selectively bind antigen (CD86), including but not limited to single domain antibodies (also referenced in the art as sdAbs, dAbs, and/or nanobodies) and VHH domains based on the heavy chains of camelids.
  • CD86 single domain antibodies
  • sdAbs, dAbs, and/or nanobodies VHH domains based on the heavy chains of camelids.
  • the extracellular domain as disclosed herein comprises an antigen binding region that may be an antigen-binding region comprising or consisting of the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:24.
  • the antigen-binding region may alternatively comprise or consist of a SEQ ID NO:1 or SEQ ID NO:24 variant amino acid sequence, which variant amino acid sequence is defined herein as having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:1 or SEQ ID NO:24, respectively, and is further characterized by specific binding to CD86.
  • the SEQ ID NO:1 or SEQ ID NO:24 variant amino acid sequence has at least 85% sequence identity to SEQ ID NO:1 or SEQ ID NO:24, respectively (and, again, exhibits CD86 specific binding activity).
  • the antigen binding region may alternatively comprise or consist of a fragment of SEQ ID NO:1 or SEQ ID NO:24, or a fragment of a SEQ ID NO:1 or SEQ ID NO:24 variant amino acid sequence, which fragment is characterized by specific binding to CD86.
  • the antigen binding region may also comprise a humanized version of the antigen binding domain of SEQ ID NO:1 or SEQ ID NO:24, characterized by specific binding to CD86.
  • Specific binding activity to CD86 is preferably tested via recombinant protein binding assays, whether cell or polypeptide based, as known in the art.
  • specific binding activity to CD86 is measured in the context of the CAR as expressed in a T cell by assessing T cell activation in response to the antigen (i.e., T cell activation on binding to CD86).
  • increasing concentrations of Fc or HIS-tagged recombinant CD86 protein are coated on a plate and incubated overnight at 4°C. Following blocking and washing, CAR-transduced T cells are added to the plate and T cell activation is measured with flow cytometry as known in the art or described herein.
  • Increased T cell activation correlating with increasing concentration of the CD86 protein indicates specific binding activity of the CAR for CD86. It is preferred that the extracellular domain of the CAR, and/or a component part thereof, does not exhibit binding activity for one or more Fc receptors. It is preferred that the extracellular domain, and/or a component part thereof, does not exhibit binding to one or more of, preferably all of, an IgG receptor (e.g. FcyRI, FcyRI IA, FcyRI IB, FcyRI IC, FcyRI HA, FcyRI II B, FcyRIV, and/or FcRn), an IgE receptor (e.g. FCERI and/or FCERI I), an IgM receptor (e.g.
  • an IgG receptor e.g. FcyRI, FcyRI IA, FcyRI IB, FcyRI IC, FcyRI HA, FcyRI II B, FcyRIV, and/or F
  • FcpR FcpR
  • an IgA/IgM receptor e.g. plgR, FcaRI and/or FcapR
  • Binding to one or more Fc receptor can be determined by the skilled person using any method known in the art or described herein.
  • the extracellular domain may further comprise additional regions, e.g., a peptide spacer connecting the antigen binding region to the transmembrane domain of the CAR.
  • the optional peptide spacerwithin the extracellular domain of the CAR of the invention comprises a flexible amino acid sequence connecting the antigen binding region to the transmembrane domain.
  • the flexible spacer allows the antigen-binding region to orient in different directions to facilitate ligand recognition and binding. It is preferred that the spacer region in the optional peptide spacer does not promote secondary structures and/or does not adopt three- dimensional structures. It is further preferred that the spacer is biologically neutral (other than optionally having a tag function as described herein).
  • the spacer does not have biological activity, e.g., interact with one or more receptors or ligands endogenously expressed by the cell expressing the CAR and/or the subject to which the cell expressing the CAR is to be administered.
  • the spacer does not consist or comprise a ligand or receptor (or portion thereof) that interacts with a counterpart receptor, endogenously expressed by either (1) the cell expressing the CAR of the invention or (2) the subject to be administered the T cell of the invention.
  • the optional spacer as described herein may comprise a hinge region as is known in the art or described herein. Any extracellular part of a protein comprising an extracellular domain, e.g., as provided among others by the CD nomenclature, may be used as a hinge domain in the extracellular domain of the CAR of the invention.
  • Exemplary spacers include, without limitation, a CD8 hinge domain, a CD28 hinge domain, a TLR5 hinge domain and a CD86 linker domain.
  • Any hinge domain known in the art or described herein can be used in the disclosed CARs and in the practice of the disclosed methods, including hinge domains from non-human or human proteins.
  • the spacer comprises or consists of a human hinge domain. It is most preferred that, in embodiments of the CAR of the invention comprising a spacer, the spacer comprises or consists of a human CD8 hinge domain.
  • a non-limiting example of this most preferred embodiment is a CAR comprising a spacer comprising or consisting of the human CD8 hinge domain having the amino acid sequence of SEQ ID NO:3 (which may be, for example, encoded by SEQ ID NO:4).
  • the extracellular domain of the CAR of the invention (e.g., expressed by the lymphocyte of the invention) comprises a spacer
  • the spacer may further comprise a detectable tag (e.g., a peptide sequence) allowing detection and/or purification of the extracellular domain, the (expressed) CAR and/or cell expressing the CAR.
  • Suitable tags allowing detection and/or purification include but are not limited to protein tags (e.g., HIS-tag, HA-tag, c-myc-tag, FLAG-tag), bi-or polycistronic vectors containing truncated proteins (examples include but are not limited to CD19, CD20, CD34, epidermal growth factor receptor (EGFR) or intracellular or transmembrane-located fluorescent proteins (e.g., enhanced green fluorescent protein (eGFP)) (Hu and Huang, Front. Immunol. (2020); 11: 1770).
  • a preferred non-limiting example of a detectable tag is a c-myc tag.
  • the c-myc tag is a peptide derived from the c-myc gene product allowing the detection and/or purification of the polypeptide comprising it and/orthe cell expressing the polypeptide comprising the c-myc tag.
  • the c-myc tag comprises or consists of the amino acid sequence SEQ ID NO:7 (which may be, for example, encoded by the nucleic acid sequence SEQ ID NO:8).
  • a non-limiting example of this most preferred embodiment is a CAR comprising a spacer comprising or consisting of the amino acid sequence of SEQ ID NO:9 (which may be, for example, encoded by SEQ ID NQ:10).
  • the extracellular domain of the CAR of the invention preferably comprises (i) an scFv antigen binding region specific for CD86 and (ii) an optional spacer comprising a human hinge region with an optional detection/purification tag. It is most preferred that the extracellular domain of the CAR of the invention (e.g., expressed by the lymphocyte for use of the invention) comprises (i) an scFv antigen binding region specific for CD86 and (ii) a spacer comprising a human CD8 hinge region with an optional detection/purification tag. It is preferred that the optional spacer is biologically neutral (other than optionally having a tag function) as described herein.
  • a non-limiting example of the above-described most preferred embodiment of the CAR of the invention (or of the CAR recombinantly expressed by the lymphocyte of the invention and for the use of the invention) comprises an extracellular domain comprising or consisting of (A) an antigen binding region that comprises or consists of
  • the CAR of the invention (or of the CAR recombinantly expressed by the lymphocyte of the invention and for the use of the invention) comprises an extracellular domain comprising or consisting of
  • amino acid sequence of SEQ ID NO:9 may, for example, be encoded by SEQ ID NQ:10.
  • SEQ ID NQ:10 amino acid sequence of SEQ ID NO:9
  • the extracellular domain of the most preferred embodiment immediately above preferably biologically neutral as defined herein. Most preferably, said extracellular domain of the most preferred embodiment above does not have binding activity for one or more Fc receptors.
  • a further specific example of such a most preferred embodiment of the CAR of the invention explained above (or of the CAR recombinantly expressed by the lymphocyte of the invention and for the use of the invention) comprises an extracellular domain comprising or consisting of
  • the extracellular domain of the most preferred embodiment immediately above preferably biologically neutral as defined herein. Most preferably, said extracellular domain of the most preferred embodiment above does not have binding activity for one or more Fc receptors. Therefore, the invention also provides a lymphocyte recombinantly expressing a CAR comprising (i) an scFv antigen binding region specific for CD86 and (ii) an optional spacer comprising a hinge region with an optional detection/purification tag.
  • the antigen binding region of the CAR recombinantly expressed by the lymphocyte of the invention comprises or consists of SEQ ID NO:1 or SEQ ID NO:24, an amino acid sequence variant of SEQ ID NO:1 or SEQ ID NO:24, which variant is an amino acid sequence at least 85% identical to SEQ ID NO:1 or SEQ ID NO:24, respectively, or a fragment of SEQ ID NO:1 or SEQ ID NO:24, or a fragment of the sequence variant of SEQ ID NO:1 or SEQ ID NO:24, which variant or fragment is characterized by specifically binding CD86.
  • the extracellular domain of the CAR expressed by the lymphocyte of the invention comprises both an antigen-binding region (that may be a murine, human or humanized scFv (or variant amino acid sequence or fragment as described above)) and a spacer comprising a human hinge region and a detection/purification tag.
  • an antigen-binding region that may be a murine, human or humanized scFv (or variant amino acid sequence or fragment as described above)
  • a spacer comprising a human hinge region and a detection/purification tag.
  • (C) a fragment of the amino acid sequence of (i) or (ii) characterized by specifically binding CD86 and having a c-myc tag.
  • amino acid sequence of SEQ ID NO:11 may, for example, be encoded by SEQ ID NO:12.
  • amino acid sequence of SEQ ID NO:26 may, for example, be encoded by SEQ ID NO:27.
  • the extracellular domain of the most preferred embodiment immediately above preferably biologically neutral as defined herein. Most preferably, said extracellular domain of the most preferred embodiment above does not have binding activity for one or more Fc receptors.
  • the CAR of the invention comprises, in addition to the extracellular domain, a transmembrane domain.
  • the transmembrane domain can be any transmembrane domain known in the art or described herein suitable for use in such in a recombinant CAR, e.g., suitable for use in a signaling protein which signal is elicited by binding of the extracellular domain to the target antigen.
  • Suitable transmembrane domains can be readily selected by the skilled person based on routine methods and knowledge in the art. It is most preferred that the transmembrane domain be of human origin, e.g., the human CD28 transmembrane domain (SEQ ID NO:5).
  • the CAR of the invention (as may be comprised by the lymphocyte of the invention) comprises, in addition to the extracellular domain and transmembrane domain, also an intracellular domain having T cell activating activity.
  • the intracellular domain having T cell activating activity (alternately referenced as an intracellular T cell activating domain) may comprise one or more stimulatory domains that transduce the signals necessary for lymphocyte (e.g., T cell) activation on the binding of the extracellular region to target antigen.
  • cytoplasmic signaling domains are known in the art and include, for example, but not limited to, the intracellular signaling domain of CD3 , CD28, 4-1BB, 0X40, as well as combinations thereof.
  • the intracellular T cell activating domain of the CAR as described herein, or of the CAR recombinantly expressed by the lymphocyte as described herein preferably comprises the signaling domain of the human CD3 chain and/or at least one costimulatory domain that is an intracellular domain of a human endogenous T cell receptor.
  • a costimulatory domain may be an intracellular domain of at least CD28 but is not limited to this specific example.
  • the intracellular signaling domain may comprise multiple costimulatory domains, for example not only including the signaling domains of the CD3 chain and CD28, but also of, e.g., CD137(4- 1BB) as is known in the art.
  • the intracellular T cell activating domain of the CAR as described herein or the CAR expressed by the lymphocyte as described herein comprises the signaling domain of the human CD3 chain and a costimulatory domain comprising an intracellular domain of human CD28 as is known in the art.
  • Sequences of the signaling domain of the human CD3 chain are known and include, but are not limited to SEQ ID NO:17 (which may be encoded by SEQ ID NO:18); similarly sequences of suitable human CD28 co-stimulatory domains are also known and include, but are not limited to SEQ ID NO:19 (which may be encoded by SEQ ID NQ:20).
  • the CAR of the invention or the CAR recombinantly expressed by the lymphocyte as described herein (i.e., for use in the treatment of cancer characterized by the expression of CD86), may comprise
  • (C) an intracellular domain comprising an intracellular T cell activating domain and/or at least one co-stimulatory domain that transduces the signals necessary for lymphocyte (e.g., T cell) activation on the binding of the extracellular region to target antigen.
  • lymphocyte e.g., T cell
  • the CAR of the invention or the CAR recombinantly expressed by the lymphocyte as described herein (i.e., for use in the treatment of cancer characterized by the expression of CD86), most preferably comprises
  • an extracellular domain comprising (i) an antigen binding region that is an scFv specific for CD86, a variant amino acid sequence of such an scFv specific for CD86, or a fragment of such scFv or variant sequence which fragment is specific for CD86; and
  • Non-limiting examples of such a CAR comprise or consist of the amino acid sequence of SEQ ID NO:13 (which may, for example, be encoded by SEQ ID NO: 14) or SEQ ID NO:28 (which may, for example, be encoded by SEQ ID NO: 29).
  • the CAR as described herein or the CAR expressed by the lymphocyte as described herein alternatively comprises or consists of a variant amino acid sequence of SEQ ID NO:13 or SEQ ID NO:28.
  • Such a variant amino acid sequence can be an amino acid sequence variant polypeptide having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:13 or SEQ ID NO:28, respectively, provided that the sequence variant is characterized by specifically binding to CD86, exhibits the c-myc tag and exhibits T cell activating activity on binding to CD86.
  • T cell activating activity can be tested through release of pro-inflammatory cytokines e.g., I FNy, IL-2, TNFalpha or GM- CSF) or the up-regulation of T cell activation or exhaustion markers (including but not limited to CD69, CD25, 4-1BB, CD28, PD-1, LAG-3 and Tim3) in response to CD86 binding.
  • pro-inflammatory cytokines e.g., I FNy, IL-2, TNFalpha or GM- CSF
  • T cell activation or exhaustion markers including but not limited to CD69, CD25, 4-1BB, CD28, PD-1, LAG-3 and Tim3
  • the CAR as described herein or the CAR expressed by the lymphocyte as described herein may also comprise or consist of a fragment of the amino acid sequence of SEQ ID NO:13 or SEQ ID NO:28 or of a fragment of the variant amino acid sequence of SEQ ID NO:13 or SEQ ID NO:28, provided that such fragment is characterized by specifically binding to CD86, by exhibiting the c-myc tag and by exhibiting T cell activating activity on binding to CD86.
  • transgenic proteins to be localized in the cell membrane require a signaling sequence for membrane localization, which signaling sequence is encoded by a nucleic acid sequence operably linked to the nucleic acid sequence encoding the membrane protein.
  • the signaling sequence is translated together with the membrane protein, but is subsequently cleaved in post-translational processing.
  • Such signal proteins are well known in the art, and can be suitable selected to localize, e.g., CARs comprising SEQ ID NO:13, their variant sequences and/or functional fragments as explained herein to the membrane using routine and standard methods.
  • Exemplary amino acid sequences comprising SEQ ID NO:13 together with suitable membrane localization sequences include SEQ ID NO:15 (which may be encoded, for example, by SEQ ID NO:16).
  • SEQ ID NO:28 includes such suitable membrane localization sequence MALPVTALLLPLALLLHAARP, which is subsequently cleaved via post-translational processing. Accordingly, the present invention extends to a CAR represented by SEQ ID NO:28 of which the membrane localization sequence has been removed.
  • the specific binding to CD86 may be any binding that is accomplished between the target protein CD86 and the CAR as described herein, or between the target protein CD86 and the lymphocyte recombinantly expressing the CAR as described herein. It is furthermore understood that the T cell activating activity on binding to CD86 as described herein is provided by the CAR or the lymphocyte recombinantly expressing the CAR.
  • the CAR as disclosed herein is characterized in that (i) the specific binding is considered to be the specific binding of a lymphocyte recombinantly expressing the CAR to CD86; and/or (ii) that the T cell activating activity on binding to CD86 is determined in a lymphocyte recombinantly expressing the CAR as described herein.
  • the specific binding as well as the T cell activating activity on binding to CD86 may be determined by methods known in the art and/or as described herein. Non-limiting examples of suitable methods are further disclosed in Xu et. al. (Methods Mol Biol. (2020); 2108:159- 165).
  • the present invention provides a CAR and a lymphocyte recombinantly expressing a CAR as described herein for use in the treatment of cancer. Accordingly, provided is a genetically engineered lymphocyte for use as a medicament.
  • CD86 may or may not be expressed by the cancer cells (i.e., the diseased cells) themselves.
  • a cancer also remains characterized by the expression of CD86 where it is not expressed by the cancerous or diseased cells themselves, but where it is expressed by cells resident within the cancer/disease parenchyma and which are not cancer or disease cells.
  • Such cells resident in the cancer/tumor/disease parenchyma that are not disease cells but that may express CD86 include, but are not limited to, tumor resident immune cells or tumor infiltrating immune cells such as macrophages. As demonstrated in the Examples, CD86 was found highly expressed across all subtypes of polarized tumor-infiltrating macrophages, contrary to the literature which described CD86 to be mainly expressed on anti-tumorigenic Ml macrophages ((Berta ni FR et al., Scientific Reports (2017);7:8965, Jablonski KA et al., PLoS One (2015);10(12):e0145342)).
  • the invention also provides a lymphocyte recombinantly expressing a CAR for use in the treatment of cancer characterized by the presence of polarized tumor-associated macrophages expressing CD86.
  • Presence is understood to describe cancer in which tumor-associated macrophages represent a component of the tumor microenvironment.
  • the cancer targeted by the CAR, or the lymphocyte expressing the CAR as described herein may be any cancer including solid tumors known in the art such as breast cancer or pancreatic cancer, but is preferably a hematological cancer.
  • Hematological cancers are understood in the art as cancers that initiate in blood-forming tissue, such as the bone marrow, or in the cells of the immune system. Nonlimiting examples thereof include leukemia, lymphoma, and multiple myeloma. It is furthermore considered that precancerous disorders such as "preleukemic" blood disorders including myelodysplastic syndrome (MDS) or myeloproliferative neoplasms (MPN) are encompassed by the invention.
  • MDS myelodysplastic syndrome
  • MPN myeloproliferative neoplasms
  • the present inventors have identified CD86 as a ubiquitous target characterizing specific hematological cancers. Accordingly, the cancer characterized by the expression of CD86 targeted by the lymphocyte recombinantly expressing the CAR as described herein is most preferably acute myeloid leukemia (AML), Hodgkin's lymphoma (HL) and non-Hodgkin's lymphoma (NHL).
  • AML acute myeloid leukemia
  • HL Hodgkin's lymphoma
  • NHL non-Hodgkin's lymphoma
  • NHL non-Hodgkin's lymphoma
  • NHL non-Hodgkin's lymphoma
  • HL Hodgkin's lymphoma
  • cHL classical Hodgkin's lymphoma
  • NLPHL very rare nodular lymphocyte predominant Hodgkin's lymphoma
  • it is preferred that the HL is cHL.
  • the cancer may be relapsed or refractory cancer.
  • the relapsed or refractory cancer is a hematological cancer as described herein.
  • the term "relapsed” means that the cancer has returned in the absence of any therapy following successful initial therapy.
  • the term “refractory” means that the cancer is progressing despite active therapy.
  • relapsed or refractory cancer is understood as cancer which has been/is treated with a tumor-targeting agent (non-limiting examples include anti-cancer drugs, antibodies and CAR T cells) other than anti-cancer treatments which directly target or interfere with the antigen CD86.
  • Exemplary anti-cancer treatments which have been described as to may result in a relapse of disease or refractory cancer are anti-CD30 CAR therapy and anti-CD33 CAR therapy, respectively.
  • the relapsed or refractory cancer is preferably a relapsed or refractory hematological cancer. More preferably, the relapsed or refractory cancer is relapsed or refractory AML, HL or NHL.
  • the relapsed cancer is most preferably CD30-negative relapse of HL and the refractory cancer is most preferably CD33-refractory AML.
  • the present invention also encompasses the treatment of hematological cancer (preferably AML, HL and/or NHL) using the lymphocyte expressing the CAR as described herein, the CAR as described herein and/or using one or more CD86 targeting agents.
  • a targeting agent may be any molecular entity which specifically interferes with, targets, and/or binds to CD86 expressed by the cancer, tumor resident cells, or cancer/disease parenchyma, e.g. immune cells present in the tumor microenvironment such as polarized tumor-associated macrophages expressing CD86.
  • targeting agents include but are not limited to small molecule inhibitors targeting the CD86 downstream signaling and CD86-blocking antibodies.
  • the treatment of hematological cancer can be effected by the lymphocyte expressing the CAR alone, by the one or more CD86 targeting agents alone, or by a combination thereof.
  • combination treatment includes administering the CAR lymphocyte of the invention concurrently with the one or more additional CD86 targeting agents as well as administration of the CAR lymphocyte prior or subsequent to the one or more additional CD86 targeting agents. When administered concurrently, they may be in the same or different preparations.
  • the invention further provides a polynucleotide encoding the CAR as described herein.
  • polynucleotide which generally relates to a nucleic acid sequence/nucleic acid molecule and is furthermore defined herein in detail.
  • the polynucleotide encoding the CAR as disclosed herein can be part of a vector but is not limited thereto.
  • the present invention therefore also relates to a vector comprising such a polynucleotide encoding the CAR as described herein.
  • the vector is used as vehicle to artificially carry the genetic material encoding the CAR of the invention into a host cell.
  • the vector comprising a polynucleotide as described herein may be any suitable vector, however, in the present invention, the vector is preferably a retroviral vector, an expression vector or a retroviral expression vector.
  • the vector comprising a polynucleotide encoding the CAR of the invention is preferably (i) a retroviral vector, and/or (ii) an expression vector.
  • Both the polynucleotide as well as the vector described herein are recognized as nucleic acid molecules which preferably replicate autonomously in a host cell (e.g. in a transduced cell) into which it has been introduced. Therefore, the invention further relates to a host cell comprising the polynucleotide encoding the CAR or the vector comprising such a polypeptide as disclosed herein, whether a lymphocyte (e.g., T cell) or not.
  • lymphocyte e.g., T cell
  • cells other than lymphocytes may suitably be used as host cells comprising the nucleic acid and/or vector of the invention for the purpose, e.g., of amplifying the nucleic acids and/or vectors.
  • Any cell suitable for genetic modification may be used as host cell.
  • Suitable host cells are known in the art and include primary cells as well as cell lines.
  • the host cell comprising the polynucleotide or the vector as disclosed herein is preferably a lymphocyte.
  • the present invention further provides a host cell comprising the polynucleotide or the vector (comprising the polynucleotide) as described herein, which is a lymphocyte expressing the CAR as described herein.
  • the host cell of the invention is a T cell, NK cell or innate lymphoid cell and expresses the CAR as described herein.
  • a host cell comprising the polynucleotide or the vector encoding such a CAR, and/or the lymphocyte recombinantly expressing the CAR, their use, as well as the methods for their production are provided not only as therapeutic tools but will also be understood to have applicability as model systems for investigating disease therapies.
  • a lymphocyte in the context of the present invention may be any lymphocyte known in the art, known or believed to be of use in an in vitro or in vivo model system.
  • lymphocyte recombinantly expressing the CAR as described herein i.e., for use in the treatment of cancer characterized by the expression of CD86
  • host cell is preferably a human lymphocyte as described herein
  • the invention encompasses host cells, e.g., lymphocytes of other mammalian species known to be of use in model systems, including but not limited to cells of rodent, canine, feline, porcine, caprine, ovine and primate origin.
  • the cells of the invention are primary human lymphocytes (e.g., including but not limited to NK cells and T cells), and most preferably primary human T cells (e.g., including but not limited to CD3+ T cell, a CD8+ T cell, a CD4+ T cell, a y6 T cell, an invariant T cell or a NK T cell).
  • primary human T cells e.g., including but not limited to CD3+ T cell, a CD8+ T cell, a CD4+ T cell, a y6 T cell, an invariant T cell or a NK T cell.
  • the invention also encompasses induced pluripotent stem cell (iPSC)-derived T cells, genetically engineered lymphocytes that are derived from lymphocyte cell lines (whether of human or non-human origin) and genetically engineered lymphocytes that are primary cells of human or non-human origin.
  • iPSC induced pluripotent stem cell
  • the lymphocytes of the invention recombinantly expressing the CAR of the invention may either be a directly genetically engineered lymphocyte, i.e., a lymphocyte that has been directly subjected to genetic engineering methods, or may be a lymphocyte derived from such a lymphocyte, e.g., a daughter cell or progeny of a lymphocyte that was directly genetically engineered.
  • the genetically engineered lymphocyte of the invention may be a directly genetically engineered lymphocyte as well as any cell derived therefrom, such as a daughter cell obtained by culture of the directly engineered/modified lymphocyte.
  • Lymphocytes recombinantly expressing the CAR as described herein are envisioned for use in therapy and may be a lymphocyte autologous to the patient to be treated (i.e., the donor from which the cells were derived and recipient are the same subject) or alternatively a lymphocyte allogenic to the patient to be treated (i.e., the donor from which the cells were derived is different from the recipient). Where the cells are allogenic, they may be further genetically engineered or prepared such that they are not alloreactive.
  • lymphocytes have been engineered (e.g., genetically engineered) such that they are rendered incapable of reacting to/recognizing allogenic (foreign) cells (in particular, other than those expressing the target antigen of the CAR).
  • the genetically engineered lymphocytes of the invention can be additionally or alternatively engineered so as to prevent their own recognition by the recipient's immune system. Lymphocytes can be rendered non-alloreactive and/or incapable of eliciting or being recognized by an immune system by any means known in the art or described herein.
  • non-alloreactive cells can comprise genetic modifications to reduce or eliminate expression of the endogenous T cell receptor (TCR) genes or the endogenous TCR.
  • TCR T cell receptor
  • the lymphocyte or host cell recombinantly expressing the CAR as described herein i.e., for use in the treatment of cancer characterized by the expression of CD86
  • the genetic modification to the lymphocyte to reduce or eliminate alloreactivity and/or to reduce or eliminate self-antigen presentation as known in the art or as described herein, e.g., the reduction or elimination of expression of the endogenous T cell receptor (TCR) alpha or beta chain genes, or of the endogenous TCR can be performed before, concurrently with, or subsequent to the genetic engineering to express the CAR as described herein.
  • TCR T cell receptor
  • the lymphocytes or host cell recombinantly expressing the CAR as disclosed herein may also be genetically engineered to further express recombinant constructs including DNR, CD40- CD40L, KO Lag3, Tim3, PD-1 or desired fusion receptors but not limited thereto.
  • the lymphocytes or host cell recombinantly expressing the CAR as disclosed herein may also be genetically engineered to further recombinantly express an exogenous cytokine receptor which may be adjuvant, e.g., in selecting, maintaining, expanding or stimulating the desired (primary) cell/cell population.
  • interleukin-2 receptor IL-2R
  • interleukin-7 receptoror interleukin-15 IL-15R
  • the lymphocytes of the present invention may be further genetically modified according to none, one, two or all of the following: modified to reduce or eliminate expression of the endogenous T cell receptor (TCR) alpha or beta chain genes; modified to exhibit reduced or eliminated expression of the endogenous TCR; modified to recombinantly express an exogenous cytokine receptor; modified to reduce or eliminate alloreactivity; and/or modified so that it does not elicit an immune response or cannot be recognized by the recipient's immune system.
  • TCR T cell receptor
  • the terms “does not elicit an immune response”, “cannot be recognized by the recipient's immune system”, “immunologically neutral” and/or analogous terms are not to be understood as absolutes.
  • Cells engineered for such activity (or lack of activity) may exhibit some immunologic activating/stimulating activity, but at reduced levels relative to the levels of a control cell prior to the relevant modifications, e.g., genetic engineering.
  • Inhibition of immune stimulatory activity or determination of immune response can be performed according to any method known in the art or described herein.
  • the present invention further provides a method for the production of a lymphocyte expressing the CAR as described herein.
  • the lymphocyte to be produced may be the lymphocyte of the invention recombinantly expressing a CAR for use in the treatment of cancer characterized by the expression of CD86 as disclosed herein, or may be the host cell comprising the genetic information to express the CAR as disclosed herein.
  • the method for production comprises the steps of (i) introducing into the lymphocyte or host cell the polynucleotide encoding the CAR or the vector comprising the polynucleotide encoding CAR (e.g., an expression vector), (ii) culturing the lymphocyte or host cell recombinantly engineered according to (i) under conditions allowing the expression of the CAR; and (iii) recovering the engineered lymphocyte or host cell.
  • the invention also encompasses a genetically engineered lymphocyte or host cell expressing the CAR of the invention obtainable by the methods as disclosed herein.
  • the methods disclosed herein also encompass methods for expanding lymphocytes or host cells after the genetic engineering for expression of the CAR (and optional further genetic modifications as disclosed herein) as well as lymphocytes and host cells obtained after such expansion.
  • the genetically engineered lymphocytes or host cells may be expanded by any suitable method known in the art or described herein.
  • methods accomplishing such expansion include exposure to one or more of the following: exposure to anti-CD3 antibodies, to anti-CD28 antibodies, and to one or more cytokines.
  • the lymphocyte is a T cell e.g., a human T cell and most preferably a primary human T cell
  • the expansion is be performed at least by exposure to one or more suitable cytokines such as interleukin-2 (IL-2) and/or interleukin-15 (IL-15).
  • suitable cytokines such as interleukin-2 (IL-2) and/or interleukin-15 (IL-15).
  • the present invention further provides the genetically engineered lymphocyte recombinantly expressing the CAR or the lymphocyte obtainable by the method as disclosed herein within a pharmaceutically acceptable carrier in the form of a pharmaceutical composition.
  • a pharmaceutical composition as disclosed herein comprises genetically engineered lymphocytes allogenic to the subject to be treated, such lymphocytes can be further genetically modified to be non-alloreactive and/or incapable of being recognized by the recipient's immune system as is known in the art or described herein.
  • lymphocytes may be further genetically modified to reduce or eliminate expression of the endogenous T cell receptor (TCR) alpha or beta chain genes, or to exhibit reduced or eliminated expression of the endogenous TCR, and/or may be modified to recombinantly express an exogenous cytokine receptor.
  • TCR T cell receptor
  • the invention also relates to the following items:
  • CD86 Cluster of Differentiation 86
  • said extracellular domain comprises an antigen binding region that is an scFv specific for said CD86.
  • a CAR comprising an extracellular domain that specifically binds CD86, a transmembrane domain, and an intracellularT cell activating domain, said extracellular domain comprising an antigen binding region that is an scFv antigen binding region specific for CD86 and said intracellularT cell activating domain comprising the signaling domain of the CD3 chain and/or at least one costimulatory domain that is an intracellular domain of an endogenous T cell receptor.
  • SEQ ID NO:13 or SEQ ID NO:28 (a) the amino acid sequence of SEQ ID NO:13 or SEQ ID NO:28; (b) an amino acid sequence that is at least 85% identical to SEQ ID NO:13 or SEQ ID NO:28, (a SEQ ID NO:13 or SEQ ID NO:28 variant amino acid sequence), wherein said SEQ ID NO:13 or SEQ ID NO:28 variant amino acid sequence is characterized by specifically binding to CD86, by having a c-myc tag and further characterized by having T cell activating activity when expressed by a lymphocyte and on binding to CD86; or
  • (c) a fragment of the amino acid sequence of (a) or (b), wherein the fragment is characterized by specifically binding to CD86, by having a c-myc tag and further characterized by having T cell activating activity when expressed by a lymphocyte and on binding to CD86.
  • the CAR according to any one of items 3 to 8 for use in the treatment of cancer.
  • the lymphocyte for use according to any one of items 1, 2, and 4 to 8, or the CAR for use according to item 9, wherein the cancer is refractory or relapsed cancer.
  • AML acute myeloid leukemia
  • Non-Hodgkin's lymphoma (NHL).
  • the lymphocyte for use according to any one of items 1, 2, 4 to 7, and 10 to 12, the CAR according to any one of items 3 to 8, or the CAR for use according to any one of items 9 to 12, wherein said specific binding is the specific binding of a lymphocyte recombinantly expressing said CAR to CD86.
  • a vector comprising the polynucleotide according to item 14.
  • a host cell comprising the polynucleotide according to item 14 or the vector according to item 15.
  • the host cell according to item 16 which is a T cell, NK cell or innate lymphoid cell and expresses the CAR according to any one of items 3 to 8.
  • step (c) recovering the engineered lymphocyte; wherein said method comprises optionally expanding said lymphocyte in step (b) and/or subsequent to step (c) by exposure to one or more of an anti-CD3 antibody; an anti-CD28 antibody; and one or more cytokines that is at least interleukin-2 (IL-2) or interleukin-15 (IL-15).
  • IL-2 interleukin-2
  • IL-15 interleukin-15
  • Figure 1 Workflow of computational CAR target antigen identification by stepwise evaluation against a set of criteria for an ideal and effective CAR target antigen.
  • a total of 12 different, publicly available scRNA-seq datasets were used for the analysis (544,764 sequenced single cells). Number of screened genes are illustrated at the bottom.
  • scRNA-seq single-cell RNA-sequencing
  • HSPC hematopoietic stem and progenitor cells
  • CSPA Cell surface protein atlas
  • HPA Human protein atlas.
  • Figure 3 Volcano plot showing the identified target antigens with their respective -loglO p-value and Iog2 average fold change from differential expression analysis between neoplastic HRS cells and their healthy counterpart as a control. Left: genes overexpressed on reactive lymph node (RLN)-derived B cells. Right: Genes overexpressed on HRS cells.
  • RNN reactive lymph node
  • Figure 4 Flow cytometric analysis of the expression of the respective target antigens on a panel of cHL cell lines (L-428, L-540, KM-H2) or Nalm-6 control cells. Absolute antigen density was measured with quantitative flow cytometric analyses as described. Shown ist the fold change of the absolute molecule count of antibody stained versus isotype control staining. Dark grey indicates cHL-cell lines, light grey indicates staining on Nalm-6 cells, p-values are based on two-way ANOVA (mixed-effects analysis). The significance was considered as: p ⁇ 0.05 (*), p ⁇ 0.01 (**), p ⁇ 0.001 (***) and p ⁇ 0.0001 (****) for all comparisons.
  • FIG. 5 Expression of CD86 determined by FACS analysis. CD86 expression on AML cell lines THP-1, Mv4-ll, OCI-AML3, PL-21, MOLM-13, U937. Acute lymphoblastic leukemia (ALL) cell line Nalm-6 was used as negative control. Representative FACS plot of at least three independent experiments is shown. Black curve indicates antibody staining and light grey line indicates isotype control.
  • Figure 6 Percentage of CD86+ cells on primary AML samples compared to an isotype control determined by flow cytometry. Pooled results ⁇ s.e.m from a total of 12 patients are depicted.
  • Figure 7 Expression of CD86 determined by FACS analysis. CD86 expression on cHL cell lines L-428, L-540 or KM-H2. ALL cell line Nalm-6 was used as negative control.
  • Figure 8 Chip cytometric analysis of of primary FFPE cHL tissue. Expression of CD4, CD86, CD30, CD20 and DNA stain was carried out respectively. HRS cells were identified by morphology. Representative pictures demonstrating the expression of CD86 on HRS in primary cHL tissue.
  • A) LR lymphocyte rich subtype.
  • B) MC mixed cell ula rity subtype.
  • C) NS nodular sclerosis subtype (NS).
  • Figure 9 Microarray analysis of a dataset generated by Brune V et al., J Exp Med (2008);205(10):2251-68. Heatmap depicts expression of CD86 or well- characterized target antigens CD19, CD20 and CD22 on B-NHL malignancies (right) or control B cells (left).
  • FL follicular lymphoma; BL, Burkitt lymphoma; DLBCL, diffuse large B cell lymphoma, RLN, reactive lymph node
  • Figure 11 Effect of anti-CD86-CAR T cells on AML (A) or cHL (B) cell lines.
  • GFP-expressing AML cell lines TFP-1, MV4-11, OCI-AML, PL-21, MOLM-13 or U937 cells or GFP- expressing cHL cell lines (L-428, L-540, KM-H2) were cocultured with transduced T cells expressing CD86-CAR.
  • CD33 CAR, CD30 CAR or CD19 CAR were used as control.
  • Antigen negative Nalm-6 cells were used as controls.
  • T cell activation as determined by IL-2 release quantified by ELISA.
  • FIG. 12 T cell proliferation of CD86 CAR T cells determined by FACS analysis.
  • CD86, CD33 or CD19 CAR T cells were stained with a proliferation dye prior to co-cultures with AML cell lines or Nalm-6 control cells. Top two rows, CD86 CAR, middle two rows, CD33 CAR, bottom two rows CD19 CAR. Proliferation was determined by trace dilution after 7 days. Representative FACS plots of three independent donors are shown.
  • FIG. 13 T cell proliferation of CD86 CAR T cells determined by FACS analysis.
  • CD86, CD33 or CD19 CAR T cells were stained with a proliferation dye prior to co-cultures with cHL cell lines L-428 (A), L-540 (B) or KM-H2 (C) or Nalm-6 cells (D).
  • Proliferation was determined by trace dilution after 7 days. Representative FACS plots of three independent donors are shown.
  • FIG. 14 Target specificity of CD86 CAR T cells were determined by FACS analysis. CD86 or CD19 CAR T cells (CTRL-transduced) were incubated on Fc-immobilized recombinant human CD86 protein at the indicated dose. 2% BSA was used as a negative control. T cell activation indicated by upregulation of CD69 on T cells was measured after 24 hours using flow cytometry. Shown are pooled results ⁇ s.e.m of three independent donors.
  • FIG. 15 Therapeutic effect of CD86-CAR T cells on AML cell compared to CD33 or CD19 CAR T cells.
  • Transduced T cells expressing CD86-CAR, CD33-CAR or CD19-CAR were cocultured with AML cell lines THP-1 (A) and MV4-11(B) or antigen negative Nalm-6 cells (C) at the indicated effector to target cell ratios (E:T ratios).
  • Specific cell lysis was quantified using BioGio Luciferase assay. Shown are pooled results ⁇ s.e.m of three independent donors.
  • Figure 16 Therapeutic effect of CD86-CAR T cells on cHL cell lines compared to CD30 or CD19 CAR T cells.
  • Transduced T cells expressing CD86-CAR, CD33-CAR or CD19- CAR were cocultured with cHL cell lines L-428 (A), L-540 (B) or KM-H2 (C) at the indicated effector to target cell ratios (E:T ratios).
  • Specific cell lysis was quantified 1 using BioGio Luciferase assay. Shown are pooled results ⁇ s.e.m of three independent donors.
  • Figure 17 Therapeutic effect of CD86-CAR T cells on NHL cell lines compared to CD30 or CD19 CAR T cells.
  • Transduced T cells expressing CD86-CAR, CD30-CAR or CD19- CAR were cocultured with NHL cell lines Z-138 (A), HBL-1 (B) or Raji (C) at the indicated effector to target cell ratios (E:T ratios).
  • Specific cell lysis was quantified using BioGio Luciferase assay. Shown are pooled results ⁇ s.e.m of three independent donors, p-values are based on two-way ANOVA (mixed-effects analysis). The significance was considered as: p ⁇ 0.05 (*), p ⁇ 0.01 (**), p ⁇ 0.001 (***) and p ⁇ 0.0001 (****) for all comparisons.
  • FIG. 18 Therapeutic effect of CD86-CAR T cells on primary AML blasts compared to CD33 or untransduced T cells (UT).
  • Transduced T cells expressing CD86-CAR or CD33- CAR or UT were cocultured with primary AML blasts of different donors at the indicated effector to target cell ratios (E:T ratios). Specific cell lysis was quantified using flow cytometry. Shown are pooled results ⁇ s.e.m of three seven independent AML patients.
  • FIG 19 In vivo therapeutic efficacy of CD86-CAR T cells in human xenograft AML mouse models.
  • AML was established in mice by intravenous injection of the human AML cell line MV4-11 expressing luciferase.
  • Transduced T cells expressing CD86-CAR, CD33-CAR or CD19-CAR were intravenously injected after tumor establishment.
  • FIG. 20 In vivo therapeutic efficacy of CD86-CAR T cells in human xenograft cHL mouse models.
  • cHL was established in mice by intravenous injection of the human cHL cell line L-540 expressing luciferase.
  • Transduced T cells expressing CD86-CAR, CD30-CAR or CD19-CAR were intravenously injected after tumor establishment.
  • FIG. 21 In vivo therapeutic efficacy of CD86-CAR T cells in human xenograft cHL mouse models.
  • cHL was established in mice by intravenous injection of the human cHLcell line L-428 expressing luciferase.
  • Transduced T cells expressing CD86-CAR, CD30-CAR or CD19-CAR were intravenously injected after tumor establishment.
  • A p-values are based on two-way ANOVA (mixed-effects analysis).
  • B Statistical significance was calculated using log-rank test.
  • A, B The significance was considered as: p ⁇ 0.05 (*), p ⁇ 0.01 (**), p ⁇ 0.001 (***) and p ⁇ 0.0001 (****) for all comparisons.
  • Figure 22 In vivo therapeutic efficacy of CD86-CAR T cells in human patient-derived xenograft (PDX) AML mouse models. AML was established in mice by intravenous injection of the human PDX-573 expressing luciferase. Transduced T cells expressing CD86-CAR, CD33-CAR or CD19-CAR were intravenously injected after tumor establishment.
  • PDX patient-derived xenograft
  • A p-values are based on two-way ANOVA (mixed-effects analysis).
  • B Statistical significance was calculated using log-rank test.
  • A, B The significance was considered as: p ⁇ 0.05 (*), p ⁇ 0.01 (**), p ⁇ 0.001 (***) and p ⁇ 0.0001 (****) for all comparisons.
  • Figure 23 In vivo therapeutic efficacy of CD86-CAR T cells in human patient-derived xenograft (PDX) NHL mouse models.
  • NHL was induced in recipient animals through i.v. injection of NHL PDX cells.
  • Transduced T cells expressing CD86-CAR or untransduced T cells were intravenously injected after tumor establishment (2 weeks after tumor injection, presence of tumor cells detected by FACS in sentinel animals). Mice were sacrificed after 55 days and tumor load was quantified by FACS.
  • Spleen weight as a sensitive marker for tumor load was measured after sacrificing the mice.
  • B Flow cytometric measurement of the tumor load in spleen, bone marrow or blood of the sacrificed mice.
  • Figure 24 In vitro generated macrophages were generated as described and polarized towards Ml-like (top), M2-like (middle) or Mh-like (bottom) macrophages. Phenotype and expression of CD86 was analyzed by flow cytometry.
  • A Flow cytometric staining for CD163 and CD206.
  • B Expression of CD86 on different in v/tro-polarized macrophages. Shown are representative flow cytometric images of at least three independent donors.
  • Figure 25 In vitro polarized macrophages (Ml-like (top), M2-like (middle) or Mh-like (bottom)) macrophages were co-cultured with proliferation-dye stained CD86-CAR, CD30-CAR or CD19 CAR T cells for a total of 7 days.
  • A Lysis of macrophages determined by flow cytometry.
  • B T cell activation was measured by ELISA.
  • Figure 26 In vivo therapeutic efficacy of CD86-CAR T cells in human patient-derived xenograft (PDX) AML mouse models. AML was established in mice by intravenous injection of the human PDX-388 expressing luciferase. Transduced T cells expressing CD86-CAR, CD33-CAR or CD19-CAR were intravenously injected after tumor establishment.
  • PDX patient-derived xenograft
  • A p-values are based on two- way ANOVA (mixed-effects analysis).
  • B Statistical significance was calculated using log-rank test.
  • A, B The significance was considered as: p ⁇ 0.05 (*), p ⁇ 0.01 (**), p ⁇ 0.001 (***) and p ⁇ 0.0001 (****) for all comparisons.
  • FIG. 27 In vivo therapeutic efficacy of CD86-CAR T cells in human xenograft cHL mouse models.
  • cHL was established in mice by intravenous injection of the human cHLcell line L-428-CD30 KO expressing luciferase.
  • Transduced T cells expressing CD86-CAR, CD30-CAR or CD19-CAR were intravenously injected after tumor establishment.
  • Figure 28 Off-tumor expression of CD80, CD86, PD-L1 and CD30 was analyzed by scRNA- seq.
  • Single-cell atlas consisting of publicly available scRNA-seq datasets used for determination gene expression patterns was comprised of 266 different specimens, 11 different organs and over 2.5 million sequenced single cells.
  • Figure 29 CD86 expression in comparison to well-described AML-associated antigens IL3RA (CD123) or CD33 using single cell sequencing. Sequencing data from 16 different AML patients after sequencing a total of 30.712 cells.
  • Figure 30 (A) Expression of CD86 or CD33 on CD34+ cord blood-derived hematopoietic stem cells (HSC) from healthy donors as determined by FACS analysis. HSC were stained after expansion for a total of 7 days as described in the methods section. Total frequency of CD86 and CD33 expressing cells on live hematopoietic stem cells (identified after gating on fixable viability dye-negative cells). Shown are pooled results ⁇ s.e.m of 2 to 3 independent donors.
  • HSC cord blood-derived hematopoietic stem cells
  • Off-target killing of CD86- CAR and CD33-CAR T cells was determined by coculturing transduced T cells expressing CD86-CAR or CD33-CAR with bone marrow-derived CD34+ stem cells from healthy human donors. Shown are representative results from three independent donors. Target cell lysis was determined by quantification of viable cells using flow cytometry. Untransduced T cells (UT) were used as control. Shown are pooled results ⁇ s.e.m of 2 to 3 independent donors, p-values are based on two-way ANOVA (mixed-effects analysis). The significance was considered as: p ⁇ 0.05 (*), p ⁇ 0.01 (**), p ⁇ 0.001 (***) and p ⁇ 0.0001 (****) for all comparisons.
  • Figure 31 (A) Binding affinity of developed anti-CD86 antibodies against full-size recombinant CD86 protein or an CD86 epitope (peptide 7072). (B) Binding affinity of developed anti-CD86 antibodies against full-size recombinant CD86 protein or an CD86 epitope (peptide 7072). Clone 3E11 was further subcloned (into 3E11-R2-2D5, herein termed 2D5 and 3G8).
  • Figure 32 List of sequence identifiers and corresponding sequences.
  • T cells are already established as major target structures and effectors in oncology, and first clinical trials demonstrate that T cell-based therapies are a promising approach for the treatment of a variety of human diseases including malignant conditions.
  • CAR T cells as well as bispecific antibodies against CD33 are under investigation. However, they have been shown to yield clinically to severe side-effects, likely due to a lack of specificity of CD33 as target structure.
  • CD30-redirected immunotherapies are clinically shown to provide effectiveness in HL (Ramos et al., J Clin Oncol (2020);38:3794-3804); however, early reports have demonstrated tumor escape, attributed to antigen-negative disease relapse after anti-CD30 CAR T cell therapy (Kim & Vega, Blood (2022);139:951). Similarly, such antigen-negative disease relapse is described for CAR T cell-based NHL treatment (Majzner R.G. and Mackall C.L., Nat Med. (2019),25(9):1341-1355.; Majzner R.G. and Mackall C.L., Cancer Discov. (2016);8(10):1219-1226; Sterner R.C. and Sterner R.M., Blood Cancer Journal (2021), 11:69; Xu et al., Front. Immunol. (2021), 11-2020).
  • CD86 is a type I membrane protein that is a member of the immunoglobulin superfamily. CD86, along with CD80, provides costimulatory signals necessary for T cell activation and survival. CD86 and CD80 bind as ligands to costimulatory molecule CD28 on the surface of all naive T cells, and to the inhibitory receptor CTLA-4 (cytotoxic T-lymphocyte antigen-4). CD28 and CTLA-4 have important, but opposite roles in the stimulation of T cells. Binding to CD28 promotes T cell responses, whereas binding to CTLA-4 inhibits T cell responses.
  • CTLA-4 cytotoxic T-lymphocyte antigen-4
  • CD86 was known to be expressed on subpopulations of hematologic cancer, however previously, CD86 was in particular known for its role in the CD80-CD86-CTLA-4-CD28 axis which was primarily investigated for its immuno-modulatory functions (Van Gool et al., Leukemia (1997);11:846- 851)). Therapeutic efforts on the CD80-CD86-CTLA-4-CD28 axis have been made by examining treatment efficacy of CTLA-4-T cells, however, these cells were described to potentially present a risk for clinical development even in the autologous setting (Lin et al, Front. Immunol. (2021);12: Art. 642528).
  • CD86 is a broadly expressed target structure in AML, HL and nHL that can be effectively used as a target molecule in ACT.
  • lymphocytes genetically engineered to express an anti-CD86 chimeric antigen receptor (anti-CD86 CAR), improve therapeutic efficacy in adoptive therapeutic strategies.
  • the methods disclosed herein are applicable to any type of lymphocyte capable of being used in adoptive therapy, including, but not limited to, natural killer (NK) cells and T cells.
  • T cells of use in accordance with the methods disclosed herein include, for example, CD4+ T cells, CD8+ T cells, and y6 T cells.
  • the present invention provides a lymphocyte recombinantly expressing a chimeric antigen T cell receptor (CAR) for use in the treatment of cancer characterized by the expression of cluster of differentiation (CD86).
  • a lymphocyte preferably a human lymphocyte, more preferably a primary human lymphocyte and most preferably a primary human T cell, NK cell or innate lymphoid cell that has been genetically engineered to recombinantly express an anti-CD86 CAR.
  • the lymphocytes according to the present invention can be any lymphocyte described herein or known in the art to be suitable for use, in particular, in an adoptive cell therapy.
  • ILCs innate lymphoid cells
  • T cells NK cells
  • non-cytotoxic ILCs innate lymphoid cells
  • ILCs are understood as the innate system counterpart of T cells. Although ILCs lack a T cell receptor, they exhibit the capacity to induce cell death (e.g., by means of the TRAIL pathway) and secrete cytokines similarly to T cells.
  • ILCs are subclassified into ILC1, ILC2, and ILC3 which share similarities with T cell subsets Thl, Th2 and Thl7, respectively.
  • ILCs are tissue resident cells than can rapidly respond to diverse environmental signals and show a remarkable plasticity. The plasticity and their ability to migrate to and reside within different tissues separately and/or in combination lead to their therapeutic advantages, e.g., for use in the treatment of solid tumors.
  • the lymphocytes may also be applicable for uses outside of therapies, such as in screening methods and/or in model systems, e.g., of use in in vitro assays or in vivo animal models. Therefore, the invention also encompasses the use of non-human sequences in the development of the CARs, genetically engineered non-human lymphocytes and/or genetically engineered lymphocytes derived from cell lines or induced pluripotent stem cells (iPSC), which may be of human or non-human origin.
  • iPSC induced pluripotent stem cells
  • Exemplary sequences that may be of use in this respect include hinge domains as explained herein of human origin, e.g., a human CD8 hinge domain comprising or consisting of SEQ ID NO:3 (which may be encoded, for example, by SEQ ID NO:4).
  • a human CD8 hinge domain comprising or consisting of SEQ ID NO:3 (which may be encoded, for example, by SEQ ID NO:4).
  • transmembrane and intracellular (T cell activation) sequences may also be used in this respect.
  • Exemplary such sequences include human transmembrane domains (such as a human CD28 transmembrane domain (e.g., SEQ ID NO:5, which may be encoded by SEQ ID NO:6)), human intracellular T cell activating domains (such as the intracellular T cell activation domain from human CD3 (e.g., SEQ ID NO:17, which may be encoded by SEQ ID NO:18)), and human intracellular T cell co-stimulatory domains (such as the stimulatory domain of human CD28 (e.g., SEQ ID NO:19, which may be encoded by SEQ ID NO:20).
  • human transmembrane domains such as a human CD28 transmembrane domain (e.g., SEQ ID NO:5, which may be encoded by SEQ ID NO:6)
  • human intracellular T cell activating domains such as the intracellular T cell activation domain from human CD3 (e.g., SEQ ID NO:17, which may be encoded by SEQ ID NO:18)
  • the iPSCs may be originally derived from any suitable cell but are preferably developed into T cells (T-iPSCs).
  • T-iPSCs T cells
  • lymphocytes include NK cells, inflammatory T lymphocytes, cytotoxic T lymphocytes, helper T lymphocytes, CD4+ T lymphocytes, CD8+ T lymphocytes, y6 T lymphocytes, invariant T lymphocytes and NK T lymphocytes.
  • the genetically engineered lymphocyte i.e., the lymphocyte recombinantly expressing the CAR as described herein, is a genetically engineered human lymphocyte.
  • the cell of the invention is a genetically engineered human NK cell or T cell, more preferably a primary human NK or T cell, and most preferably a primary human T cell, which may be, e.g., a CD8+T cell, a CD4+-T cell, or y6 T cell.
  • primary and analogous terms in reference to a cell or cell population as used herein correspond to their commonly understood meaning in the art, i.e., referring to cells that have been obtained directly from living tissue (i.e., a biopsy such as a blood sample) or from a subject, which cells have not been passaged in culture, or have been passaged and maintained in culture but without immortalization. It is more preferred that the engineered lymphocytes are engineered primary human lymphocytes. Primary cells have undergone very few population doublings, if any, subsequent to having been obtained from the tissue sample and/or subject, and are therefore more representative of the main functional components and characteristics of in situ tissues and cells as compared to continuous tumorigenic or artificially immortalized cell lines.
  • the primary lymphocytes described herein can be isolated and/or obtained from a number of tissue sources, including but not limited to, peripheral blood mononuclear cells isolated from a blood sample, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and/or tumors by any method known in the art or described herein.
  • tissue sources including but not limited to, peripheral blood mononuclear cells isolated from a blood sample, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and/or tumors by any method known in the art or described herein.
  • a genetically engineered primary T cell of the present invention is that having been obtained and/or isolated from a T cell population from subject (preferably a human patient).
  • lymphocytes including T cells
  • Methods for isolating/obtaining specific populations of lymphocytes (including T cells) from patients or from donors include as a first step, for example, isolation/obtaining a donor or patient sample known or expected to contain such cells, e.g., a blood or bone marrow sample.
  • the desired cells e.g., NK cells orT cells, are separated from the other components in the sample.
  • Methods for separating a specific population of desired cells from the sample include, but are not limited to, e.g., leukapheresis for obtaining T cells from the peripheral blood sample from a patient or from a donor; isolating/obtaining specific populations from the sample using a FACSort apparatus; and selecting specific populations from fresh biopsy specimens comprising living lymphocytes by hand or by using a micromanipulator (see, e.g., Dudley et al., Immunother. (2003), (26):332-342; Robbins et al., Clin. Oncol. (2011), (29):917- 924; Leisegang, J. Mol. Med. (2008), (86):573-58).
  • fresh biopsy specimens refers to a tissue sample (e.g., a tumor tissue or blood sample) that has been or is to be removed and/or isolated from a subject by surgical or any other known means.
  • the isolated/obtained cells are subsequently cultured and expanded according to routine methods known in the art for maintaining and/or expanding the desired primary cell and/or primary cell population.
  • culture may occur in the presence of an anti-CD3 antibody; in the presence of a combination of anti-CD3 and anti-CD28 monoclonal antibodies, and/or in the presence of an anti-CD3 antibody, an anti-CD28 antibody and one or more cytokines, e.g., interleukin-2 (IL-2) and/or interleukin-15 (IL-15) (see, e.g., Dudley et al., Immunother. (2003), (26):332-342; Dudley et al., Clin. Oncol. (2008), 26:5233-5239).
  • IL-2 interleukin-2
  • IL-15 interleukin-15
  • lymphocytes or T cells which methods are also encompassed by the invention.
  • methods include but are not limited to isolation and culture of primary cell sub-populations, e.g., primary T cell sub-populations such as CD3+, CD28+, CD4+, CD8+, and y6, as well as the isolation and culture of other primary lymphocyte populations such as NK T cells or invariant T cells.
  • selection methods can comprise positive and/or negative selection techniques, e.g., wherein the sample is incubated with specific combinations of antibodies and/or cytokines to select for the desired sub-population.
  • the skilled person can readily adjust the components of the selection medium and/or method and length of the selection using well known methods in the art. Longer incubation times may be used to isolate desired populations in any situation where there is or are expected to be fewer desired cells relative to other cell types, e.g., such as in isolating tumor infiltrating lymphocytes (TIL) from tumor tissue or from immunocompromised individuals.
  • TIL tumor infiltrating lymphocytes
  • multiple rounds of selection can be used in the disclosed methods. Enrichment of the desired population is also possible by negative selection, e.g., achieved with a combination of antibodies directed to surface markers unique to the negatively selected cells.
  • cell sorting and/or selection via negative magnetic immunoadherence or flow cytometry which use a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected can be used.
  • a monoclonal antibody cocktail typically including antibodies specific for CD14, CD20, CDllb, CD16, HLA-DR, and CD8 is used.
  • the methods disclosed herein also encompass removing T regulatory cells, e.g., CD25+ T cells, from the population to be genetically engineered. Such methods include using an anti-CD25 antibody, or a fragment thereof, or a CD25-binding ligand, such as IL-2.
  • the lymphocyte recombinantly expressing the CAR as described herein may be a genetically engineered autologous primary lymphocyte.
  • autologous refers to any material isolated, derived and/or obtained from the same individual to whom it is later to be reintroduced, e.g., in the context of an autologous adoptive therapy, such as autologous adoptive T cell therapy (ACT) wherein the same individual is both the donor and recipient.
  • ACT autologous adoptive T cell therapy
  • the genetically engineered lymphocyte may be a genetically engineered autologous primary lymphocyte, including but not limited to a genetically engineered primary autologous NK cell or a primary autologous T cell, such as a primary autologous CD8+ T cell, a primary autologous CD4+ T cell, a primary autologous y6 T cell, a primary autologous invariant T cell or a primary autologous NK T cell.
  • a genetically engineered primary autologous NK cell including but not limited to a genetically engineered primary autologous NK cell or a primary autologous T cell, such as a primary autologous CD8+ T cell, a primary autologous CD4+ T cell, a primary autologous y6 T cell, a primary autologous invariant T cell or a primary autologous NK T cell.
  • the methods and materials disclosed herein are not limited to autologous lymphocytes isolated and/or derived from the subject to be subsequently treated with the lymphocytes (and/or are not limited to the use of such autologous lymphocytes, e.g., as a medicament in the treatment of a disease characterized by CD86).
  • the methods disclosed herein also encompass the use and production of genetically engineered allogeneic lymphocytes, in particular primary lymphocytes.
  • an "allogeneic lymphocyte” is a lymphocyte (e.g., a T cell) isolated from a donor of the same species as the recipient but not genetically identical to the recipient.
  • Such allogenic cells can be used in adoptive therapies without or, preferably, with further modification as described herein, e.g., to reduce or inactivate the allogenic reactions in the intended recipient by the engineered T cell to the host (e.g., graft versus host reactions) as well as those immune reactions of the host against the engineered T cell (e.g., host versus graft reactions).
  • modifications can be made by any method known in the art and/or described herein (such cells are known in the art and referenced herein as "non-alloreactive" lymphocytes/T cells).
  • the cells may be further genetically engineered or prepared such that they are not alloreactive.
  • not alloreactive indicates that the lymphocytes/T cells have been engineered (e.g., genetically engineered) such that they are rendered incapable of reacting to/recognizing allogenic (foreign) cells other than the cells expressing the target antigen specifically bound/recognized by the antigen-binding region of the CAR of the invention. Therefore, non-alloreactive cells derived from third-party donors may become universal, i.e., recipient independent.
  • the genetically engineered lymphocytes of the invention can be additionally or alternatively engineered so as to rendering them incapable of eliciting an immune response and/or of being recognized by the recipient's immune system, preventing them from being rejected.
  • Such cells that are non-alloreactive and/or that are incapable of eliciting an immune response or being recognized by the recipient's immune system may also be termed "off-the-shelf" lymphocytes as is known in the art.
  • Lymphocytes can be rendered non-alloreactive and/or incapable of eliciting or being recognized by an immune system by any means known in the art or described herein.
  • the lymphocytes of the invention may have disruption or deletion of the endogenous major histocompatibility complex (MHC).
  • MHC major histocompatibility complex
  • Such cells may have diminished or eliminated expression of the endogenous MHC when compared to an unmodified control cell, preventing or diminishing activation of the recipient's immune system against the autologous cells.
  • non-alloreactive cells can have reduced or eliminated expression of the endogenous T cell receptor (TCR) when compared to an unmodified control cell.
  • TCR T cell receptor
  • Such non-alloreactive T cells may comprise modified or deleted genes involved in self-recognition, such as but not limited to, those encoding components of the TCR including, for example, the alpha and/or beta chain.
  • the genetic modifications to reduce or eliminate alloreactivity i.e., to render the cell non-alloreactive other than against cells expressing the antigen of choice (i.e., that specifically bound by the antigen-binding region of the CAR of the invention)
  • reduce or eliminate self-antigen presentation i.e., so as to prevent them from eliciting an immune response or being recognized by the recipient's immune system
  • self-antigen presentation i.e., so as to prevent them from eliciting an immune response or being recognized by the recipient's immune system
  • non-alloreactive/off the shelf lymphocytes can be obtained from a repository and then engineered to express the CAR of the invention according to the methods described herein and subsequently used in the treatment, in particular, of cancers characterized by CD86.
  • the modifications to render the lymphocyte non-alloreactive and/or incapable of eliciting an immune response and/or being recognized by the recipient's immune system were performed prior to the genetic engineering to express the CAR.
  • the donor and/or recipient of the lymphocytes as disclosed herein may be any living organism in which an immune response can be elicited (e.g., mammals).
  • Examples of donors and/or recipients as used herein include humans, dogs, cats, mice, rats, monkeys and apes, as well as transgenic species thereof, and are preferably humans.
  • the term "recombinantly expressing" and analogous terms refers to (i) a cell that has been recombinantly/genetically modified to express a CAR as described herein; as well as (ii) the progeny of such a cell that maintains expression of such a polypeptide, e.g., obtainable by culture of the originally modified cell.
  • Methods of genetically engineering cells to express polypeptides of interest are well known and routine in the art and include methods of introducing nucleic acids encoding the polypeptide in an appropriate form (e.g., in an expression vector) into cells via chemical or viral means.
  • a cell "recombinantly expressing" a polypeptide according to the invention generally encompasses the deliberate introduction of a nucleic acid molecule into the cell so that it will express the introduced sequence/molecule to produce a desired substance, e.g., a CAR.
  • "Recombinantly expressing” encompasses any means of introducing the nucleic acid sequence or molecule (e.g., a polynucleotide or vector) into the cell described herein or known in the art suitable to allow expression of the encoded polypeptide.
  • transduction methods commonly understood to refer to the introduction of a foreign nucleic acid into a cell using a vector, including the use of a viral vector
  • transfection methods commonly understood to refer to the introduction of a foreign nucleic acid into a cell using non-viral means such as chemical- or electric poration, microinjection, etc.
  • “recombinantly expressing” in more general terms also encompasses methods of transformation, i.e., the introduction of a gene, DNA or RNA sequence into a host cell, such that the host cell will express the introduced gene or sequence to produce a desired substance, such as a polypeptide (e.g., a CAR) encoded by the introduced gene or sequence (e.g., a polynucleotide sequence).
  • a polypeptide e.g., a CAR
  • the introduced gene or sequence can be referenced as a “cloned”, “foreign”, or “heterologous” gene or sequence, or a "transgene”.
  • the introduced nucleic acid molecule/sequence can also comprise additional heterologous sequences including, for example, heterologous promoters, start, stop, promoter, signal, secretion, or other sequences used by a cell's genetic machinery operatively linked to the coding sequences described herein, as well as further regulatory nucleic acid sequences well known in the art and/or described herein.
  • the introduced gene or sequence can include nonfunctional sequences or sequences with no known function. According to the methods disclosed herein, a host cell that receives and expresses introduced DNA or RNA has been "genetically engineered".
  • genetically engineered in the context of the methods and products described herein is equivalent to transformed, transduced and/or transfected, and the genetically engineered cell is, for example, a transformant or a clone and is "transgenic".
  • the DNA or RNA introduced to the host cell i.e., the lymphocyte, can be derived from any source, including cells of the same genus or species as the host cell, or cells of a different genus or species.
  • the lymphocytes recombinantly expressing the CAR of the invention are preferably cultured under controlled conditions outside of their natural environment.
  • the term "culturing” as used herein indicates that the engineered cells are maintained in vitro.
  • the genetically engineered lymphocytes are cultured under conditions allowing the expression of the CAR as described herein. Conditions that allow the maintenance of lymphocytes and expression of a desired transgene therein are commonly known in the art and include but are not limited to culture in the presence of agonistic anti-CD3- and anti-CD28 antibodies, as well as one or more cytokines such as interleukin 2 (IL-2), interleukin 7 (IL-7), interleukin 12 (IL-12) and/or interleukin 15 (IL-15).
  • IL-2 interleukin 2
  • IL-7 interleukin 7
  • IL-12 interleukin 12
  • IL-15 interleukin 15
  • lymphocyte recombinantly expressing the CAR as described herein comprising the steps of modifying (e.g., transducing) the cell to express the CAR, culturing the modified/recombinant cell under conditions allowing the expression of the CAR, and recovering said genetically engineered cell.
  • the lymphocytes as described herein may be activated and/or expanded as is known in the art.
  • methods according to the invention may also include a step of activating and/or expanding a primary lymphocyte or lymphocyte population.
  • such methods can encompass culturing the cells with appropriate agents such as agents that activate stimulatory receptors (e.g., agonistic antibodies) and/or target ligands of endogenous or recombinant receptors as routine in the art.
  • Said cells can also be expanded by co-culturing with tissue or cells expressing target ligands of endogenous or recombinant receptors, including in vivo, for example in the subject's blood after administrating the cells to the subject.
  • the lymphocyte recombinantly expressing the CAR may comprise a polynucleotide molecule, or a vector comprising the polynucleotide molecule, encoding the CAR as described herein.
  • the CAR of the invention comprises an extracellular domain that specifically binds CD86, a transmembrane domain, and an intracellular T cell activating domain. As such, only a part of the receptor is accessible from the intracellular space.
  • the encoded CAR i.e., the extracellular part thereof
  • the encoded CAR is expressed on the surface of the engineered cell and can be detected either directly, e.g., by flow cytometry or microscopy using antibodies specific for the CAR as described herein or a portion thereof (e.g., specific of the tag within the spacer of the extracellular domain, in particular, a c-myc tag) or indirectly, e.g., by assessing the engineered cells for anti-CD86 activity by any method known in the art and/or described herein.
  • binding to is interchangeable with the term “interacting with” and “specific for” and not only relates to a linear epitope but may also relate to a conformational epitope, a structural epitope or a discontinuous epitope consisting of two regions of the, e.g., human, target molecules or parts thereof.
  • CAR constructs that bind to the (poly)peptide/protein of interest, i.e., CD86, but that do not or do not essentially bind to any other (poly)peptide/protein expressed by the same tissue as the (poly)peptide/protein of interest, e.g., by the tumor cells, are considered specific for the (poly)peptide/protein of interest as is known and accepted in the art.
  • Methods to determine binding may comprise, inter alia, binding studies, blocking and competition studies with structurally and/or functionally closely related molecules.
  • Non-limiting examples of methods to assess specificity to CD86 include Western Blots, ELISA-, RIA-, ECL-, IRMA-tests and peptide scans.
  • Binding studies also comprise FACS analysis, surface plasmon resonance (SPR, e.g., with BIAcore), analytical ultracentrifugation, isothermal titration calorimetry, fluorescence anisotropy, fluorescence spectroscopy or by radiolabeled ligand binding assays. Furthermore, physiological assays like cytotoxic assays may be performed. Accordingly, examples for the specific interaction of an antigen-interaction-site with a specific antigen comprise the specificity of a ligand for its receptor or vice versa. Said definition particularly comprises the interaction of ligands which induce a signal upon binding to its specific receptor.
  • the term “specifically binds”, “recognizes”, “interacts with” and analogous terms designate the degree to which an antigen binding region discriminates between two antigens. This is because it is known that no antigen binding region, e.g., an antibody antigen binding region, has absolute specificity, in the sense that it will react with only one epitope whatever the conditions. That is, where other (non-target) antigens are present, an antigen binding region may react to some extent with similar epitopes on these other (non-target) antigens. However, the affinity of an antigen binding region for its target epitope/antigen is significantly greater than its affinity for related epitopes.
  • This difference in affinity is used to establish assay conditions, under which an antigen binding region binds almost exclusively to a specific (target) epitope.
  • the binding (or non-binding) of an antigen binding region to an antigen are not understood as absolutes. That is, the CAR of the invention, the cell expressing a CAR of the invention, and/or the antigen-binding region ofthe CAR ofthe invention may exhibit some (residual) binding activity for other (non-)targets, but at significantly reduced levels relative to the binding activity for CD86.
  • the antigen-binding domain of the CAR of the invention, the CAR and/or cell expressing the CAR exhibit at least 10 fold, at least 20 fold, preferably at least 50 fold, and more preferably at least 100 fold better affinity for CD86 as compared to the affinity for a non-target antigen.
  • the extracellular domain of the CAR of the invention comprises an antigen binding region specific for CD86 and a spacer.
  • the spacer is most preferably a peptide spacer which connects the antigen-binding region with the transmembrane domain of the CAR as described herein.
  • Spacers offer the advantage of allowing the different domains/regions of the CAR (i.e., the antigen binding region and the transmembrane domain of said CAR) to fold independently and exhibit the expected activity.
  • the extracellular domain, the transmembrane domain and the intracellular T cell activating domain of the CAR may be comprised in a single-chain multi-functional polypeptide.
  • the spacer as described herein is biologically neutral as defined herein, e.g. does preferably not have binding activity for one or more Fc receptors (FcR).
  • the one or more Fc receptor may be a FcRn and/or an Fey receptor as known in the art or described herein, e.g., in humans the family includes FcyRI (CD64) including isoforms FcyRla, FcyRIb and FcyRIc; FcyRII (CD32) including isoforms FcyRlla (including allotype H131 and R131), FcyRI I b (including FcyRllb-1 and FcyRI lb-2), and FcyRI Ic; and FcyRI II (CD16) including isoform FcyRllla (including allotype V158 and F158) and FcyRlllb (including allotype FcyRlllb-NAl and FcyRI II b-NA2).
  • Impairment or prevention of binding to FcRs by the spacer domain as disclosed herein prevents FcR-expressing cells from recognizing and destroying, or unintentionally activating the CAR-expressing cells, thereby minimizing or preventing immunological rejection and clearance of the therapeutically active cells.
  • Whether a CAR exhibits binding activity to an FcR can be measured by methods known to those skilled in the art including FACS, ELISA, ALPHA screen (amplified luminescent proximity homogeneous assay) or BIACORE.
  • Methods for tag detection include detection via flow cytometry or microscopy using antibodies specific for the tag, e.g., antibodies against c-myc or a portion thereof.
  • Suitable methods for purification of the CAR of the invention or of a lymphocyte recombinantly expressing the CAR as described herein are known in the art.
  • Such methods for purification include preparative chromatographic separations and immunological separations based on antigen recognition/binding (e.g., recognition or binding to CD86) and/or based on the tag regions, e.g., comprising the use of antibodies specific for c-myc or a portion thereof.
  • the CAR comprising a spacer
  • it may be derived from any extracellular part of a protein having an extracellular domain, and is preferably derived from a biologically neutral portion of such extracellular domain.
  • the spacer comprises the hinge domain of such extracellular domains, e.g., as provided among others by the CD nomenclature.
  • Such are well known in the art and include the hinge domain of CD8 and CD28.
  • the hinge domain is preferably that of CD8.
  • the hinge domain is that of human CD8, for example having the amino acid sequence as shown herein in SEQ ID NO:3 (e.g., which may be encoded, for example, by SEQ ID NO:4).
  • the extracellular domain/antigen binding region of the CAR as described herein comprises a moiety that provides specificity for CD86, and may be derived by an antibody antigen biding domain as is known in the art, e.g., in preferred embodiments, an scFv.
  • the extracellular domain/antigen binding region can be derived from human antibodies (e.g. human antibody libraries) as well as antibodies from different species as the lymphocyte donor or lymphocyte recipient, and, in this context, may optionally be chimeric or humanized, as long as the original binding activity to the target antigen is retained.
  • human antibodies e.g. human antibody libraries
  • antibodies from different species as the lymphocyte donor or lymphocyte recipient
  • a murine scFv may be equally applicable or even advantageous for CAR construction over the counterpart human or humanized scFv (see, e.g. Ramos C.A. et al., J Clin Oncol. (2020),10;38(32):3794-3804; Wutti- In Y. et al. Front Oncol. (2022),18;ll:802876).
  • the extracellular domain of the CAR as described herein is biologically neutral as defined here, e.g. does not comprise an unmodified antibody Fc domain or a part thereof, including one or more unmodified CHI, CH2, or CH3 domains, as such elements increase the risk of adverse side reactions such as FcyR binding on administration to a subject.
  • amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, y-carboxyglutamate, and O-phosphoserine.
  • Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a-carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
  • Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid but that function in a manner similar to a naturally occurring amino acid.
  • the CAR provided herein may exemplarily comprise or consist of the amino acid sequence of SEQ ID NO:13 or SEQ ID NO:28.
  • the term "functional variant" of a particular amino acid sequence encompasses variant amino acid sequences and/or fragments of the particular amino acid sequence or of the variant amino acid sequence, provided that the functional variant polypeptide exhibits or imparts the same functional activity as the particular amino acid sequence polypeptide.
  • variant amino acid sequence of a particular amino acid sequence refers to a functional polypeptide variant thereof, that does not have an amino acid sequence identical to the particular amino acid sequence, e.g., SEQ ID NO:13, but which polypeptide exhibits or imparts the same functional activity, in particular, specifically binding to CD86 and exhibiting T cell activating activity on binding to CD86, when expressed by the lymphocyte.
  • the functional variant can be any variant amino acid sequence polypeptide having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the particular amino acid sequence, e.g., SEQ ID NO:13 or SEQ ID NO:28, provided that the variant sequence is characterized by the same functional activity as the original amino acid sequence.
  • fragment of a particular amino acid sequence, e.g., of SEQ ID NO:13 or its variant amino acid sequence, refers to a functional polypeptide variant thereof that does not have an amino acid sequence identical to the particular amino acid sequence, e.g., SEQ ID NO:13 or SEQ ID NO:28, but which polypeptide exhibits or imparts the same functional activity, e.g., specifically binding to CD86.
  • the term "at least X % identical to" in connection with the amino acid sequences/polypeptides and/or the nucleic acid sequences/nucleic acid molecules/polynucleotides as used herein describes the number of matches ("hits") of identical amino acid or nucleic acid residues of two or more aligned sequences as compared to the number of residues making up the overall length of the compared sequences (or the overall compared portions thereof).
  • the percentage of residues that are the same may be determined when the (sub)sequences are compared and aligned for maximum correspondence over a window of comparison, or over a designated region as measured using a sequence comparison algorithm as known in the art, or when manually aligned and visually inspected.
  • Examples of algorithms for use in determining sequence identity include, for example, those based on CLUSTALW computer program (Thompson, Nucl. Acids Res. 2(1994), 4673-4680) or FASTA (Pearson and Lipman, Proc. Natl. Acad. Sci., 85(1988), 2444).
  • FASTA Pearson and Lipman, Proc. Natl. Acad. Sci., 85(1988), 2444
  • CLUSTALW does take sequence gaps into account in its identity calculations.
  • the BLAST and BLAST 2.0 algorithms Altschul, Nucl. Acids Res., 25(1977), 3389).
  • the BLASTP program uses as default a word length (W) of 3, and an expectation (E) of 10.
  • the BLAST program is used in methods disclosed herein.
  • the herein provided CAR e.g., the CAR recombinantly expressed by the lymphocyte provided herein, comprises a transmembrane domain.
  • Any transmembrane portion of a protein e.g., of a signal transmitting receptor can be used in the construction of the CAR.
  • proteins from which the transmembrane domain can be derived or taken include, but are not limited to, CD4, CD8 and CD28.
  • the transmembrane domain comprises or consists of a CD28 transmembrane domain.
  • Such a CD28 transmembrane domain may have an amino acid sequence of human or non-human origin. It is most preferred that the transmembrane domain used in the CAR as disclosed herein comprises or consists of the transmembrane domain of human CD28 (SEQ ID NO:5, which may be encoded, for example, by SEQ ID NO:6).
  • the CAR of the invention also comprises an intracellularT cell activating domain.
  • intracellular domains may comprise one or more stimulatory domains that transduce the signals necessary for lymphocyte (e.g., T cell) activation.
  • Such intracellular signaling domains can include, for example, but not limited to, the intracellular signaling domain of CD3 , CD27, CD28, 4-1BB, 0X40, ICOS and combinations thereof. Further, it may comprise an I L-2R
  • the intracellular T cell activating domain of the CAR as described herein, or of the CAR expressed by the lymphocyte of the invention comprises the signaling domain of the CD3 chain and/or at least one costimulatory domain that is an intracellular domain of an endogenous T cell receptor.
  • costimulatory domain can be the intracellular domain of CD28 and/or CD137(4-1BB).
  • the intracellularT cell activating domain of the CAR as described herein or the CAR expressed by the lymphocyte of the invention preferably comprises the signaling domain of the human CD3 chain and a costimulatory domain which comprises an intracellular domain of at least human CD28.
  • the activity of the stimulatory signalling region(s), which provide(s) T cell activation may be measured by the same means as determining T cell activation.
  • the invention further relates to polynucleotides encoding the CAR of the invention and to vectors comprising such a polynucleotide encoding the CAR of the invention.
  • a lymphocyte disclosed herein does not express the CAR as described herein endogenously, it is understood that such a lymphocyte has been genetically engineered so as to comprise the CAR.
  • nucleic acid sequences in accordance with the CAR, the genetically engineered lymphocyte and the methods as disclosed herein, relate to sequences of polynucleotides/nucleic acid molecules comprising purine- and pyrimidine bases.
  • nucleic acid molecule and “polynucleotide” may be interchangeably used and include DNA, such as cDNA, genomic DNA or synthetic forms of DNA, as well as RNA and mixed polymers comprising two or more of these molecules.
  • RNA as used herein comprises all forms of RNA including mRNA, tRNA and rRNA but also genomic RNA, such as in case of RNA of RNA viruses.
  • embodiments reciting "RNA" are directed to mRNA.
  • the nucleic acid molecules/nucleic acid sequences of the invention may be of natural as well as of synthetic or semi-synthetic origin.
  • the nucleic acid molecules may, for example, be nucleic acid molecules that have been synthesized according to conventional protocols of organic chemistry.
  • the person skilled in the art is familiar with the preparation and the use of such nucleic acid molecules (see, e.g., Sambrook and Russel "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Laboratory, N.Y. (2001)).
  • nucleic acid mimicking molecules known in the art such as synthetic or semi-synthetic derivatives of DNA or RNA and mixed polymers, both sense and antisense strands. They may contain additional non-natural or derivatized nucleotide bases, as will be readily appreciated by those skilled in the art.
  • nucleic acid mimicking molecules or nucleic acid derivatives include peptide nucleic acid (PNA), phosphorothioate nucleic acid, phosphoramidate nucleic acid, 2'- O-methoxyethyl ribonucleic acid, morpholino nucleic acid, hexitol nucleic acid (HNA) and locked nucleic acid (LNA), an RNA derivative in which the ribose ring is constrained by a methylene linkage between the 2' -oxygen and the 4' -carbon (see, for example, Braasch and Corey, Chemistry & Biology 8(2001), 1-7).
  • PNA peptide nucleic acid
  • HNA hexitol nucleic acid
  • LNA locked nucleic acid
  • PNA is a synthetic DNA-mimic with an amide backbone in place of the sugar-phosphate backbone of DNA or RNA, as described in, e.g., Nielsen et al., Science 254(1991), 1497; Egholm et al., Nature 365(1993), 666.
  • nucleic acid molecules may contain, for example, thioester bonds and/or nucleotide analogues. Said modifications may be useful for the stabilization of the nucleic acid molecule against endo- and/or exonucleases in the genetically engineered cell.
  • nucleic acid molecules/sequences disclosed herein may be transcribed by an appropriate vector containing a chimeric gene, which allows for the transcription of said nucleic acid molecule/sequence in the genetically engineered cell.
  • polynucleotide can be used for "gene targeting” or “gene therapeutic” approaches.
  • nucleic acid molecules/sequences are labeled. Methods for the detection of nucleic acids are well known in the art, e.g., by Southern and Northern blotting, PCR or primer extension.
  • nucleic acid molecules/sequence(s) may be a recombinantly produced chimeric nucleic acid sequence comprising any of the aforementioned nucleic acid sequences either alone or in combination.
  • the genetically engineered lymphocyte of the invention may transiently or stably express the CAR as described herein. Additionally, the expression can be constitutive or constitutional, depending on the system used as known in the art.
  • the polynucleotide or the vector encoding the polypeptide may or may not be stably integrated into the cell's genome. Methods for achieving stable integration of introduced nucleic acids encoding desired proteins are well known in the art, and the invention encompasses the use of such methods as well as those described herein.
  • the herein provided lymphocyte (most preferably a primary human T cell) or the herein provided host cell which is preferably a lymphocyte has been genetically modified by introducing the polynucleotide or the vector comprising the polynucleotide into the lymphocyte.
  • the invention encompasses vectors comprising the polynucleotide encoding the CAR as described herein.
  • the term "vector” relates to a circular or linear nucleic acid molecule that can autonomously replicate in a host into which it has been introduced.
  • the vector as used herein particularly refers to a plasmid, a cosmid, a virus, a bacteriophage and other vectors commonly used in genetic engineering as described herein or as is known in the art.
  • the disclosed vectors are suitable for the transformation of lymphocytes, preferably human lymphocytes and more preferably human primary lymphocytes, including but not limited to NK cells and T cells such as CD8+ T cells, CD4+ T cells, CD3+ T cells, y6 T cells, invariant T cells and NK T cells.
  • Vectors in connection with the present invention comprise a nucleic acid sequence, e.g., the polynucleotide as described herein, encoding the CAR of the invention.
  • the vectors of use in connection with the present invention may encode the amino acid sequence SEQ ID NO:13 or SEQ ID NO:28, or a functional variant thereof, provided that the variant is characterized by specifically binding to CD86.
  • vectors of use in connection with the present invention may also encode polypeptides comprising signaling domains to allow the proper processing and localization of the encoded polypeptide; accordingly, such vectors may encode CARs comprising membrane localization signaling peptides, e.g., as in SEQ ID NO:15.
  • the vectors disclosed herein may contain additional sequences to allow function such as replication or expression of a desired sequence in the cell system.
  • the vectors may comprise the polynucleotide encoding the CAR as described herein, under the control of regulatory sequences.
  • regulatory sequence refers to DNA sequences that are necessary to affect the expression of coding sequences to which they are operably linked.
  • control sequences generally include promoters, ribosomal binding sites, and terminators.
  • control sequences generally include promoters, terminators and, in some instances, enhancers, transactivators and/or transcription factors.
  • control sequence is intended to include, at a minimum, all components the presence of which are necessary for expression, and may also include additional advantageous components, e.g., to allow replication.
  • Regulatory or control sequences including but not limited to promoters, transcriptional enhancers and/or sequences, which allow for induced or constitutive expression of the CAR as described herein, may be employed.
  • Suitable promoters include but are not limited to the CMV promoter, the UBC promoter, PGK, the EF1A promoter, the CAGG promoter, the SV40 promoter, the COPIA promoter, the ACT5C promoter, or the TRE promoter (e.g., as disclosed in Qin et al., PLoS One.
  • the vectors of use in the present invention are preferably expression vectors. Suitable expression vectors have been widely described in the literature and the determination of the appropriate expression vector can be readily made by the skilled person using routine methods.
  • the vectors disclosed herein comprises a recombinant polynucleotide (i.e., a nucleic acid sequence encoding the CAR as described herein) as well as expression control sequences operably linked to the nucleotide sequence to be expressed.
  • the vectors as provided herein preferably further comprise a promoter.
  • the herein described vectors may also comprise a selection marker gene and a replication-origin ensuring replication in the host (i.e., a genetically engineered (e.g., transduced) lymphocyte such as a T cell).
  • the herein provided vectors may also comprise a termination signal fortranscription.
  • Between the promoter and the termination signal may be at least one restriction site or a polylinker to enable the insertion of a nucleic acid molecule encoding a polypeptide desired to be expressed (e.g., a polynucleotide encoding the CAR as disclosed herein).
  • a nucleic acid molecule encoding a polypeptide desired to be expressed e.g., a polynucleotide encoding the CAR as disclosed herein.
  • expression vectors including insertion of the encoding nucleic acid molecule/sequence and the harvest of the expressed polypeptide, is routine in the art.
  • vectors suitable for use in the present invention include cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., retroviruses) that incorporate the nucleic acid molecules encoding the CAR.
  • a viral expression vector is preferred use.
  • Methods for genetically engineering cells to express polypeptides of interest are known in the art and can generally be divided into physical, chemical and biological methods. The appropriate method for given cell type and intended use can readily be determined by the skilled person using common general knowledge.
  • Such methods for genetically engineering cells by introduction of nucleic acid molecules/sequences encoding the polypeptide of interest include but are not limited to chemical- and electroporation methods, calcium phosphate methods, cationic lipid methods, and liposome methods.
  • the nucleic acid molecule/sequence to be transduced can be conventionally and highly efficiently transduced by using a commercially available transfection reagent and/or by any suitable method known in the art or described herein.
  • mRNA transfection refers to a method well known to those skilled in the art to transiently express a protein of interest, in the present case the CAR as described herein, in a lymphocyte, e.g., a T cell. Accordingly, the methods herein may be used to genetically engineer a lymphocyte to transiently or stably (either constitutively or conditionally) express the polypeptide of interest.
  • lymphocytes may be electroporated with the mRNA coding for the CAR as described herein by using an electroporation system (such as e.g., Gene Pulser, Bio-Rad) and thereafter cultured by standard cell culture protocols (see, e.g., Zhao et al., Mol Ther. 13(2006), 151-159).
  • an electroporation system such as e.g., Gene Pulser, Bio-Rad
  • standard cell culture protocols see, e.g., Zhao et al., Mol Ther. 13(2006), 151-159.
  • Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like; see, e.g., Sambrook et al., 2012, Molecular Cloning: A Laboratory Manual, volumes 1-4, Cold Spring Harbor Press, NY.
  • Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian (e.g., human cells such as a T cells).
  • retroviral vectors are preferred for use in the methods and cells disclosed herein, viral vectors can be derived from a variety of different viruses, including but not limited to lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses; see, e.g., U.S. Pat. Nos. 5,350,674 and 5,585,362.
  • suitable retroviral vectors for transducing T cells include SAMEN CMV/SRa (Clay et al., J. Immunol. 163(1999), 507-513), LZRS-id3-IHRES (Heemskerk et al., J. Exp. Med.
  • pMP71 Engels et al., Hum. Gene Ther. 14(2003), 1155-1168
  • pGCSAM Morgan et al., J. Immunol. 171(2003), 3287-3295
  • pMSGV Zero et al., J. Immunol. 174(2005), 4415-4423
  • pMX de Witte et al., J. Immunol. 181(2008), 5128-5136.
  • Non-limiting examples of suitable lentiviral vectors for transducing T cells are, e.g., PL-SIN lentiviral vector (Hotta et al., Nat Methods. 6(2009), 370-376), pl56RRL-sinPPT-CMV- G FR-RRE/A//?el (Campeau et al., PLoS One 4(2009), e6529), pCMVR8.74 (Addgene Catalogoue No.:22036), FUGW (Lois et al., Science 295(2002), 868-872, pLVX-EFl (Addgene Catalogue No.: 64368), pLVE (Brunger et al., Proc Natl Acad Sci U S A 111(2014), E798-806), pCDHl-MCSl-EFl (Hu et al., Mol Cancer Res.
  • PL-SIN lentiviral vector Hotta et al., Nat
  • Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid- based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).
  • Other methods of state-of-the-art targeted delivery of nucleic acids are available, such as delivery of polynucleotides with targeted nanoparticles or other suitable sub-micron sized delivery system.
  • an exemplary delivery vehicle is a liposome.
  • lipid formulations is contemplated for the introduction of the nucleic acids into a host cell (in vitro, ex vivo or in vivo).
  • the nucleic acid may be associated with a lipid.
  • the nucleic acid associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid.
  • Lipid, lipid/DNA or lipid/expression vector associated compositions are not limited to any particular structure in solution.
  • Lipids may be present in a bilayer structure, as micelles, or with a "collapsed" structure. They may also simply be interspersed in a solution, possibly forming aggregates that are not uniform in size or shape.
  • Lipids may be naturally occurring or synthetic lipids. Lipids suitable for use in methods of nucleic acid molecule delivery to a host cell (i.e., to genetically engineer the host cell) can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine (“DMPC”) can be obtained from Sigma, St.
  • DMPC dimyristyl phosphatidylcholine
  • DCP dicetyl phosphate
  • Choi cholesterol
  • DMPG dimyristyl phosphatidylglycerol
  • a variety of assays may be performed.
  • Such assays include, for example, "molecular biological” assays well known to those of skill in the art such as Southern and Northern blotting, RT-PCR and PCR, "biochemical” assays such as detecting the presence or absence of a particular polypeptide, e.g., by immunological means (ELISAs and/or Western blots) or by assays described herein to identify whether the cell exhibits a property or activity associated with the engineered polypeptide, e.g., assays to assess whether the lymphocyte exhibits a desired activity such as the specific binding to CD86.
  • “molecular biological” assays well known to those of skill in the art such as Southern and Northern blotting, RT-PCR and PCR
  • biochemical assays such as detecting the presence or absence of a particular polypeptide, e.g., by immunological means (ELISAs and/or Western blots) or by assays described herein to identify whether the cell exhibits a property or activity associated with the
  • T cells are cells of the adaptive immune system that recognize their target in an antigen specific manner. These cells are characterized by surface expression of CD3 and a T cell receptor (TCR), which recognizes a cognate antigen in the context of a major histocompatibility complex (MHC). T cells may be further subdivided in CD4+ or CD8+ T cells.
  • CD4+ T cells recognize an antigen through their TCR in the context of MHC class II molecules that are predominantly expressed by antigen-presenting cells.
  • CD8+ T cells recognize their antigen in the context of MHC class I molecules that are present on most cells of the human body.
  • T cells e.g., as a culture of primary T cells
  • a cell population such as a population of peripheral blood mononuclear cells e.g., having been isolated from a patient for the purpose of autologous cell therapy
  • flow cytometry, microscopy, immunohistochemistry, RT-PCR or western blot are well known to those skilled in the art and include flow cytometry, microscopy, immunohistochemistry, RT-PCR or western blot (Kobold, J Natl Cancer Inst 107(2015), 107).
  • the genetically engineered lymphocyte of the present invention is recombinantly modified with a nucleic acid sequence/polynucleotide encoding (and driving/permitting expression of) the herein described CAR.
  • a nucleic acid sequence/polynucleotide encoding (and driving/permitting expression of) the herein described CAR In the case of cells bearing natural anti-tumor specificity (such as tumor-infiltrating lymphocytes (TIL see, e.g., Dudley et o/., J Clin Oncol. 31(2013), 2152-2159)) or antigen-specific cells sorted from the peripheral blood of patients for their tumor-specificity by flow cytometry (Hunsucker et al., Cancer Immunol Res.
  • TIL tumor-infiltrating lymphocytes
  • the genetically engineered cells described herein may only be modified to express the CAR.
  • the genetically engineered T cell of the invention may be further engineered with additional nucleic acid molecules to express, in addition to the exogenous CAR as described herein, other polypeptides of use in ACT, e.g., with a nucleic acid sequence encoding a further, exogenous, T cell receptor or a further chimeric antigen receptor (CAR) specific for a tumor of interest.
  • the T cell can be further genetically modified to disrupt the expression of the endogenous T cell receptor, such that it is not expressed or expressed at a reduced level as compared to a T cell absent of such modification.
  • both the lymphocyte or host cell for use in the methods of the invention are non-alloreactive.
  • the non-alloreactive lymphocyte or host cell is a T cell
  • such a T cell comprises genetic mutations to reduce or eliminate expression of the endogenous TCR, or of the endogenous TCR alpha or beta chain genes.
  • endogenous refers to molecules which are naturally not presented in and/or on the surface of a cell, e.g., a T cells, and which are not (endogenously) expressed in or on normal (non-transduced) cells, e.g., T cells.
  • exogenous refers to molecules which do not naturally occur in or on cells, e.g., T cells and relates to molecules which are incorporated into the cell, e.g., a T cell, which are naturally not presented in and/or on the surface of the cell and which are not (endogenously) expressed in or on normal (non-transduced) cells.
  • these artificially introduced molecules are presented in and/or on the surface of cells, e.g., T cells, after genetic engineering as accomplished by methods known in the art or as disclosed herein.
  • the term “reduced expression” and analogous terms refer to any reduction in the expression of the endogenous T cell receptor at the cell surface of a genetically modified cell when compared to a control cell.
  • the term reduced can also refer to a reduction in the percentage of cells in a population of cells that express an endogenous polypeptide (i.e., an endogenous TCR) at the cell surface when compared to a population of control cells.
  • the term “reduced expression” in connection with the expression of an endogenous T cell receptor relates to a partial knockdown, while the term “eliminated expression” relates to a complete, or essentially complete knockdown of the endogenous TCR within the population of genetically modified cells.
  • the T cell comprises genetic mutations to reduce or eliminate expression of the endogenous TCR, or of the endogenous TCR alpha or beta chain genes as described herein.
  • the lymphocyte or host cell may further recombinantly express an exogenous cytokine receptor.
  • lymphocyte or host cell expressing the CAR of the invention is of particular use in the treatment of cancer characterized by the expression of CD86 and can successfully be employed in pharmaceutical compositions.
  • pharmaceutical composition may also comprise the lymphocytes as obtained by the methods disclosed herein.
  • treatment generally mean obtaining a desired pharmacological and/or physiological effect.
  • the effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof, and/or may be therapeutic in terms of partially or completely curing the disease or condition, and/or adverse effect attributed to the disease or condition.
  • treatment covers any treatment of a disease or condition in a subject and includes: (a) preventing and/or ameliorating a proliferative disease (preferably cancer) from occurring in a subject that may be predisposed to the disease; (b) inhibiting the disease, i.e., arresting its development, such as inhibition of cancer progression; (c) relieving the disease, i.e., causing regression of the disease, such as the repression of cancer; and/or (d) preventing, inhibiting or relieving any symptom or adverse effect associated with the disease or condition.
  • the term "treatment” as used herein relates to medical intervention of an already manifested disorder, e.g., the treatment of a diagnosed cancer, in particular characterized by the expression of CD86.
  • CD86 Cluster of differentiation
  • a cancer or pre-cancer may also be characterized by the expression of CD86 where the cancer or precancerous cells do not express CD86, but where immune cells resident within the diseased tissue express CD86 e.g., infiltrating lymphocytes, in particular tumor infiltrating lymphocytes (TIL)).
  • TIL tumor infiltrating lymphocytes
  • composition can be used interchangeably with “medicament” and generally relates to a composition for administration to a patient, preferably a human patient. Furthermore, in the context of the present invention, such patient suffers from a disease characterized by the expression of CD86, wherein said disease is a malignant disease, especially a cancer of the blood.
  • the composition of the invention as described herein may also be a composition for diagnosing further comprising, optionally, means and methods for detection.
  • the pharmaceutical composition as disclosed herein may be administered locally or systematically.
  • compositions may be administered by any suitable way, including parenteral, transdermal, intraluminal, intraarterial, intrathecal administration and direct injection into the tissue or tumor, however, parenteral administrations is the preferred application method.
  • parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, orfixed oils.
  • Intravenous vehicles include fluid and nutrient replenishes, electrolyte replenishers (such as those based on Ringer's dextrose) and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like.
  • the pharmaceutical composition of the present invention might comprise proteinaceous carriers, like, e.g., serum albumin or immunoglobulins, preferably of human origin and may also comprise, optionally, suitable formulations stabilizers and/or excipients.
  • the pharmaceutical composition of the invention may comprise, in addition to the lymphocyte recombinantly expressing the CAR as described herein, further biologically active agents, depending on the intended use of the pharmaceutical composition.
  • agents may include medicaments acting on the gastro-intestinal system, cytostatic drugs, drugs preventing hyperuricemia, drugs inhibiting immunoreactions (e.g., corticosteroids), drugs acting on the circulatory system and/or agents such as T cell co-stimulatory molecules or cytokines known in the art.
  • chemotherapeutic agents include an anthracycline (e.g., doxorubicin (e.g., liposomal doxorubicin)), a vinca alkaloid (e.g., vinblastine, vincristine, vindesine, vinorelbine), an alkylating agent (e.g., cyclophosphamide, decarbazine, melphalan, ifosfamide, temozolomide), an immune cell antibody (e.g., alemtuzamab, gemtuzumab, rituximab, ofatumumab, tositumomab, brentuximab), an antimetabolite (including, e.g., folic acid antagonists, pyrimidine analogs, purine analogs and adenosine deaminase inhibitors (e.g., fluorubicin (e.g., doxorubicin (e.g., lip
  • General chemotherapeutic agents considered for use in combination therapies also include but are not limited to anastrozole, bicalutamide, bleomycin sulfate, busulfan, capecitabine, N4-pentoxycarbonyl-5-deoxy-5-fluorocytidine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, cyclophosphamide, cytarabine, cytosine arabinoside, cytarabine liposome injection, dacarbazine, dactinomycin, daunorubicin hydrochloride, daunorubicin citrate liposome injection, dexamethasone, docetaxel, doxorubicin hydrochloride, etoposide, fludarabine phosphate, 5-fluorouracil, flutamide, tezacitibine, Gemcitabine, hydroxyurea (Hydrea.RTM.), Idarubicin,
  • Anti-cancer agents of particular interest for combination with the genetically engineered lymphocyte based methods and compounds disclosed herein include: anthracyclines; alkylating agents; antimetabolites; drugs that inhibit either the calcium dependent phosphatase calcineurin or the p70S6 kinase FK506) or inhibit the p70S6 kinase; mTOR inhibitors; immunomodulators; anthracyclines; vinca alkaloids; proteosome inhibitors; GITR agonists; protein tyrosine phosphatase inhibitors; a CDK4 kinase inhibitor; a BTK inhibitor; a MKN kinase inhibitor; a DGK kinase inhibitor; or an oncolytic virus.
  • Exemplary antimetabolites include, without limitation, pyrimidine analogs, purine analogs and adenosine deaminase inhibitors): methotrexate, 5-fluorouracil, floxuridine, cytarabine, 6- mercaptopurine, 6-thioguanine, fludarabine phosphate, pentostatin, pemetrexed, raltitrexed, cladribine, clofarabine, azacitidine, decitabine and gemcitabine.
  • alkylating agents include, without limitation, nitrogen mustards, uracil mustard, ethylenimine derivatives, alkyl sulfonates, nitrosoureas, triazenes, chlormethine, cyclophosphamide, ifosfamide, melphalan, chlorambucil, pipobroman, triethylenemelamine, triethylenethiophosphoramine, temozolomide, thiotepa, busulfan, carmustine, lomustine, streptozocin, dacarbazine, oxaliplatin, temozolomide, dactinomycin, melphalan, altretamine, carmustine, bendamustine, busulfan, carboplatin, lomustine, cisplatin, chlorambucil, cyclophosphamide, dacarbazine, altretamine, ifosfamide, prednumustine, procarbazine, mechlorethamine
  • the invention further envisages the co-administration protocols with other compounds, e.g., molecules capable of providing an activation signal for immune effector cells, for cell proliferation or for cell stimulation.
  • CD86 Cluster of Differentiation 86
  • scRNA-seq Single-cell RNA-sequencing-based screening algorithms were built to identify suitable target antigens for the treatment of acute myeloid leukemia (AML).
  • Single-cell sequencing strategies in comparison to conventional bulk sequencing analysis, are able to predict expression pattern at a much higher resolution as cell-type specific expression patterns are analyzed (Zheng et al. Nat Commun (2017);8:14049).
  • HSPC hematopoietic stem and progenitor cells
  • HL Hodgkin's Lymphoma
  • scRNA-seq-based screening algorithms described above in Example 5.1.1 could not be used, since single-cell profiles of the HL-initiating tumor cells (so called Hodgkin-Reed-Sternberg cells, HRS) have not yet been obtained. This is most likely due to the fragility and the rarity of HRS cells in the tumor tissue of patients suffering from HL.
  • cHL cell lines L-428, L-540, KM-H2; control cell line: NALM-6, all purchased from DSMZ (Germany) or ATCC (USA), respectively.
  • APC anti-human FPR1 (clone: W15086B), APC anti-human CD213al (clone: SS12B), APC antihuman CD163 (clone GH1/61), anti-human CD90 (clone: 5E10), anti-human Alexa Fluor488 CD14 (clone: 63D3), anti-human CD274 (PD-L1, clone: 29E.2a3), PE anti-human CD80 (clone 2D10) and APC anti-human CD86 (clone: IT2.2).
  • Mouse IgGl (clone: MOPC-21) or mouse lgG2b (clone: 27-35) isotype controls were stained on each respective cell line (all antibodies from Biolegend, USA).
  • BD BiosciencesTM QuantibriteTM Phycoerythrin (PE) Beads were used according to manufacturer's instruction.
  • non-PE primary antibodies described above were stained with anti-mouse IgGl (clone: RMG1-1) or anti-mouse lgG2b (clone: RMG2b-l) antibodies.
  • tumor cells L-428, L-540, KM-H2 or NALM-6 control
  • KM-H2 or NALM-6 control tumor cells
  • cells were twice washed PBS and then stained on with the respective antibodies for 30 min at 4°C. Then, cells were washed twice and either analzyed as described below or stained with the respective secondary antibodies.
  • a fixable viability dye eFluorTM 780, eBioscience, USA
  • Samples were subsequently analyzed using a BD LSR FortessaTM II.
  • IGSF6 IGSF6-induced IGSF6
  • CD86 and CD30 were significantly overexpressed on all three HL lines, showing a more than 300-fold increase of expression compared to an isotype control staining (Fig. 4).
  • the other target antigens could only be detected on a small subset of HL cell lines (CD80, PD-L1) or not at all (CD14, CD90, CD163, CD213a, FPR1).
  • CD86 expression on myeloid blasts of human AML patients as well as on AML cell lines was determined using FACS analysis.
  • Human AML cell lines PL-21, THP-1, MV4-11, OCI-AML3, M0LM13, U937 were purchased from ATCC (USA).
  • NALM-6 cells were used as a negative control and were again purchased from ATCC (USA). All cell lines were cultured in RPMI containing 20% FBS, 2 mM L-Glutamine, 100 U/ml penicillin and 100 pg/ml streptomycin. Cells were grown at 37°C in a humidified incubator with 5% CO2. Short tandem repeat (STR) profiling was used to verify their origins. Cells were regularly tested for mycoplasma contamination using polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • AML blasts were obtained from the bone marrow (BM) or peripheral blood (PB) of patients suffering from acute myeloid leukemia (AML) after written informed consent in accordance with the Declaration of Helsinki and approval by the Institutional Review Board of the Ludwig-Maximilians Universitat (Munich, Germany).
  • BM bone marrow
  • PB peripheral blood
  • AML acute myeloid leukemia
  • Flow cytometric analysis was carried out using a BD LSRFortessaTM II or a Beckman Coulter CytoFLEX (LX). Flow cytometric data was analyzed using FlowJo V10.8 software. All staining steps were conducted on ice. Cells were centrifuged at 200 - 400 g for 5 min at 4°C in a precooled centrifuge. For staining of primary AML blasts and AML cell lines a maximum of 10 6 cells were counted and transferred to a U bottom 96 well plate. Cells were washed twice with ice cold phosphate-based saline (PBS) containing 2 % FBS.
  • PBS ice cold phosphate-based saline
  • CD86 was stained on ice for 30 minutes in the dark using an anti-human CD86 antibody conjugated to APC (Biolegend, Clone IT2.2), BV605 (Biolegend, IT2.2) or a purified anti-human CD86 Antibody (Biolegend, Clone IT2.2), followed by secondary staining with an anti-mouse IgGl or anti-mouse lgG2b antibody, respectively (Jackson ImmunoResearch, USA). Positive staining was validated using isotype controls (Biolegend, RMG1-1, RMG2b-l). Dead cells were excluded after staining with a fixable viability dye (eFluorTM 780, eBioscience, USA).
  • a fixable viability dye eFluorTM 780, eBioscience, USA.
  • Fig. 5 demonstrates the expression of CD86 on a panel of six different AML cell lines, while antigen-negative NALM-6 cell line did not show a positive staining.
  • BM frozen bone marrow
  • Human HL cell lines L-540, L-428 and KM-H2 were purchased from DSMZ (Germany). Cells were cultured and transduced as described in Example 5.1.3.1. 5.1.4.2. Flow cytometry
  • Example 5.1.3.3 Flow cytometric staining was carried out as described in Example 5.1.3.3. The following antibodies were used to stain for CD30 and CD19: CD30 - clone BY88 (Biolegend, US); CD19 clone HIB19 (Biolegend USA).
  • FFPE Formalin-fixed parafin-embedded
  • PE-conjugated secondary anti-rabbit antibody (Jackson ImmunoResearch, 711-116-152, 1:100) was applied for 2 h at room temperature for antibody detection. Following photobleaching for 30 s, primary antibody against CD20 (Novus Biologicals, NBP2-44743, 1:100) was applied overnight at 2 - 8°C. PE-conjugated secondary anti-mouse antibody (Jackson ImmunoResearch, 715-116-150, 1:100) was applied for 2 h at room temperature for antibody detection. Followinged by DNA-Hoechst (Invitrogen, 62249, 1:50000) staining for 5 min at room temperature, chemical bleaching was done using a quenching buffer, according to published protocols (Jarosch et.
  • CD30 staining was carried out using a primary antibody (abeam, ab23766, 1:80/1:300) after PE conjugation (PE Conjugation Kit, abeam, abl02918) for 2 h at room temperature.
  • PE conjugation Kit PE Conjugation Kit, abeam, abl02918
  • CD4 staining was conducted using primary antibody (abeam, abl33616, 1:100) conjugated to AF488 or a conjugated CD4-PE primary antibody (Novus Biologicals, NBP1-19371, 1:300, PE Conjugation Kit, abeam, abl02918). Both antibody clones were incubated for 2 h at room temperature.
  • HRS cells are derived from germinal center B cells. Besides HL, malignantly transformed germinal center B cells also give rise to various B cell-derived Non-Hodgkin Lymphomas (NHL) such as mantle cell lymphoma (MCL), follicular lymphoma (FL) and diffuse-large B cell lymphoma (DLBCL). Consequently, the expression of CD86 on various subsets of NHL was analyzed. First, the expression was analyzed using a publicly available microarray dataset generated by Brune et al. (Fig. 9).
  • NHL mantle cell lymphoma
  • FL follicular lymphoma
  • DLBCL diffuse-large B cell lymphoma
  • the datasets contained sequencing results from five primary FL samples, eleven primary DLBCL samples and five reactive lymph node (RLN) controls (Brune V et al., J Exp Med (2008);205(10):2251-68).
  • the deposited date was analzyed using a publicy available R script from Mark Dunning (tutorial concerning data from Gene Expression Omnibus from website https://sbc.shef.ac.uk/geo_tutorial/tutorial.nb.html).
  • Deposited microarray data from GSE 12453 were analyzed using the publicly available R script from Mark Dunning (REF) with minor adjustments (tutorial concerning data from Gene Expression Omnibus from website https://sbc.shef.ac.uk/geo_tutorial/tutorial.nb.html). Briefly, the series matrix file was loaded using the getGEO function of the GEOquery library. The input data were Iog2 transformed. Subsequently, only genes were kept that are expressed in more than 2 samples. The groups were defined according to the column source_name_chl of the phenotypic data of the series matrix.
  • Human cell lines K422, HBL-1, Z138, Raji and SuDHL-4 were either purchased from ATCC or gifted by third party investigators. The correct origin of the cell lines were verified by STR profiling as described in Example 5.1.3.1. Cells were cultured and transduced as described in Example 5.1.3.1.
  • FACS analysis was carried out as previously described (Example 5.1.3.3). The analysis revealed a strong expression of CD86 on various NHL cell lines compared to an isotype staining control or an antigen negative cell line (Fig. 10).
  • the following example demonstrates CD86 as targeting antigen for therapy, e.g., by using modified T-cells.
  • Example 1 revealed that CD86 was readily detected on primary AML samples as well as samples of patients suffering from various malignant lymphomas (both HL and NHL).
  • novel CAR T cells which specifically recognize CD86 were generated.
  • the following construct was designed: human-CD8alpha signal peptide - anti-CD86 VH (clone 3D1) - G4S)4-Linker- anti-CD86 VL (clone 3D1) - myc-tag - CD8 hinge- CD28 transmembrane domain -CD28 intracellular domain -CD3zeta domain (anti- CD86 CAR having the amino acid sequence of SEQ ID NO: 15).
  • the anti-CD86 single chain variable fragment was derived from a patented sequence of an anti-CD86-binding antibody (clone 3D1; Patent Number: US2002/0176855A1 (amino acid sequence SEQ ID NO:1)).
  • a myc tag was included to readily detect CAR expression.
  • Anti-CD86 CAR T cells were compared to established anti-CD19 CAR T cells (negative control for AML and HL; positive control for NHL), anti-CD30 CAR T cells (positive control, HL; negative control for NHL) and anti-CD33 CAR T cells (positive control for AML). All CAR were constructed in a similar fashion as the anti-CD86 CAR.
  • Anti-CD33 CAR T cells were designed in following configuration: CD8alpha signal peptide - anti-CD33scFv - cmyc tag - CD8 hinge- CD28 transmembrane - CD28 intracellular domain - human CD3zeta domain (anti- CD33 CAR having the nucleic acid sequence of SEQ ID NO:21).
  • the anti-CD33 scFv were designed based on the patent of the anti-CD33 antibody gemtuzumab (Appelbaum & Bernstein, Blood (2017); 130: 2373-2376).
  • Anti-CD19 CAR T cells were designed based on the patented anti-CD19-CAR-FMC63-28Z CAR T cells (WO 2015/187528 Al) (anti-CD19 CAR having the nucleic acid sequence of SEQ ID NO:22).
  • Anti-CD30 CARs were designed from patented anti-CD30 CAR T cells currently used in clinical trials (WO 2017/066122 Al) (anti-CD30 CAR having the amino acid sequence of SEQ ID NO:23).
  • T cells were isolated, cultured and transduced with either anti-CD86-CAR, anti-CD30-CAR, anti- CD19-CAR or anti-CD33-CAR as described in Example 5.2.3.
  • retroviral pMP71 (kindly provided by C. Baum, Hannover) vectors carrying the sequence of the relevant receptor were stably expressed in packaging cell lines 293Vec- Galv and 293Vec-RD114.
  • producer cell lines 293Vec-RD114-CAR-CD86, 293Vec-RD114-CAR-CD19 and 293Vec-RD114-CAR-CD30 and 293Vec-RD114-CAR-CD33 were generated. 5.2.3 T cell culture and T cell transduction
  • PBMC peripheral blood mononuclear cells
  • Isolated T cells were counted, adjusted to a cell concentration of 10 6 /ml and stimulated for 48 hours using Human T-Activator CD3/CD28 Dynabeads (Life Technologies, Darmstadt, Germany) in complete human T cell medium containing 2.5 % human Serum, 2 mM L-Glutamine, 100 U/ml penicillin, 100 pg/ml streptomycin, 1 % non-essential amino acids, 1 % Sodium Pyruvate and supplemented with recombinant human IL-2 (Peprotech, Hamburg, Germany) and IL-15 (Peprotech, Hamburg, Germany). T cell transduction was carried out by retroviral transduction.
  • Retroviral particles were generated from producer cell lines (Vec-RD114) stably expressing the desired constructs, as previously described (Example 5.2.2). Virus supernatant was added to retronektin-coated 24 well plates (12.5 pg/ml; TaKaRa Biotech, Japan) and centrifuged for 1.5 hours at 3000 g at 37° C. Following centrifugation, supernatant was removed and 10 6 pre-stimulated T cells were added to the virus-coated plates. 24 - 48 hours later T cells were removed from the plate and successful transduction was verified using flow cytometry. CAR expression was detected using fluorochrome-coupled anti-c-myc antibody (FITC, clone SH1-26E7.1.6, Miltenyi Biotec, Germany).
  • FITC fluorochrome-coupled anti-c-myc antibody
  • Human AML cell lines (THP-1, Mv4-ll, OCI-AML, PL-21, U937, MOLM-13), human HL cell lines (L-428, L-540, KM-H2) and all NHL cell lines (HBL-1, Raji, Z138) were I entivira I ly transduced to express eGFP and fLuc and cultured as described in Examples 5.1.2.1., 5.1.3.1. and 5.1.4.1.
  • Tumor cells were co-cultured with transduced T cells or untransduced control T cells at the indicated effector to target cell ratio (E:T ratio) for 48 or 72 hours, respectively. All cells were resuspended in human T cell medium that did not contain IL-2 or IL-15. CD86-negative NALM-6 cells were used as a negative control for CAR T cell-mediated killing. After 24 hours, T-cell mediated killing of cell lines were determined using either BioGioTM Luciferase Assay System (Promega Corporation, USA) or flow cytometry. Flow cytometric-based determination of tumor cell death was quantified using Count BrightTM Absolute Counting Beads (Life Technologies, Darmstadt, Germany) after gating on GFP- positive tumor cells.
  • AML blasts were thawed 3 days prior to the experiment and cultured as described in Example 5.1.3.2. On day 0 AML blasts were cocultured with allogenicT cells obtained from healthy donors. Transduced CAR T cells or control T cells were co-cultured at the indicated effector to target cells ratios (E:T ratios). 48 hours later lysis of AML blasts was determined by flow cytometry. T cells and AML blasts were grouped based on the expression of the T cell lineagea marker CD2 and the myeloid marker CD33, highly expressed on AML blasts.
  • Activation of T cells was determined by quantification of lnterleukin-2 (IL-2) or interferon gamma (IFN-y) release following co-culture of T cells and tumor cells as described above.
  • IL-2 or IFN-y levels in supernatants of co-culture experiments were measured using human IL-2 or IFN-y ELISA Kit (BD Bioscience, Germany). Measurements were carried out according to manufactures' protocol.
  • FACS antibodies were used to determine specific lysis on co-culture with primary AML blasts (Fig. 18): anti-human CD3 (clone UCHT1, Biolegend, USA), anti-human CD4 (clone OKT4 Biolegend, USA), anti-human CD8 (clone SKI Biolegend, USA), anti-human CD33 (clone P67.6, Biolegend, USA; WM53, Invitrogen/eBioscience). Samples were analyzed using Beckman Coulter CytoFLEX. As shown in Fig.
  • anti-CD86-CAR T cells showed significant activation as determined by IL- 2 or IFN-y release in the presence of human AML and HL tumor cells as compared to cultures with control-transduced T-cells, T cells alone, and/or tumor cell lines alone.
  • anti-CD86 CAR T cells did not show any signs of activation (minimal IL-2 release), illustrating the specificity of the newly developed CAR T cells.
  • CAR T cells CD86, CD19 or CD30
  • CAR T cells were stained with a proliferation dye (CellTraceTM Far Red Proliferation Dye) prior to cultures. After 7 days, proliferation of CAR T cells, indicated by dilution of the proliferation dye (thus, peak of proliferated cells is shifting to the left), was measured with flow cytometry.
  • Anti-CD86 CAR T cells showed strong proliferation in co-cultures with AML oder HL cell lines, indicated by a strong shift of the flow cytometric histograms to the left (Fig. 12 and 13). CD86 CAR T cells proliferated as strong as positive control (anti-CD33 CAR T cells, AML; anti-CD30 CAR T cells, HL).
  • T cells are specifically activated by AML cells as well as HL cells when expressing anti-CD86-CAR and that target recognition leads to sustained proliferation of anti-CD86 CAR T cells.
  • CAR T cells After 24 hours, activation of CAR T cells by plate-bound recombinant protein was measured using flow cytometry. Activation of CAR T cells was measured by upregulation of CD69 (clone FN50 Biolegend, USA) or CD107a (clone H4A3 Biolegend, USA). As seen in Fig. 14., anti-CD86 CAR T cells were specifically and dose-dependently activated by plate-bound recombinant CD86 protein (Fig. 14). In contrast, control-transduced (CD19) CAR T cells did not show any signs of activation. Furthermore, high- protein solutions (2% BSA in PBS) did not lead to activation of CD86 CAR T cells, underpinning the specificity of the CAR-mediated activation (Fig. 14).
  • anti-CD86 CAR T cells are able to lyse antigen expressing AML, HL and NHL cell lines
  • in vitro co-culture experiments were conducted as described in Example 5.2.5. All experiments were performed with fLuc-eGFP-expressing tumor cells. Tumor cell lysis was determined by luminescence measurements following target cell lysis in the presence of the fLuc substrate Luciferin as described.
  • Anti-CD86 CAR T cells showed a very high specific lysis of AML, HL and NHL tumor cells, compared to control-transduced CAR T cells (Fig. 15 to 17).
  • CD86 CAR T cells specific lysis of CD86 CAR T cells was at least comparable to anti-CD33 (for AML), anti-CD30 (for HL) or anti-CD19 CAR T cells (for NHL), illustrating the high efficacy of anti-CD86-CAR-mediated lysis (Fig. 15 to 17).
  • anti-CD86-CAR T cells Therapeutic effectivity of anti-CD86-CAR T cells was also demonstrated by determining T cellspecific lysis of primary AML blasts.
  • Primary blasts were obtained from AML patients as described above and were co-cultured with allogenic transduced T cells expressing anti-CD86- CAR, anti-CD33-CAR T cells or non-transduced control T cells. As shown in Fig. 18, anti-CD86- CAR T cells were able to specifically lyse primary AML blasts.
  • the following example demonstrates CD86 as targeting antigen as evaluated in in vivo models.
  • Tumor cells and T cells were cultured as previously described.
  • CDX cell line-derived xenograft
  • PDX patient-derived xenograft model
  • CDX cell line-derived xenograft
  • MV4-11 or human HL cell lines L-428 and L-540 served as xenograft for implantation into immunodeficient mice.
  • 10 6 MV4-11 cells or 3.5 x 10 6 L-428 or L-540 tumor cells expressing eGFP and fLuc were injected intravenously (i.v.) into immunodeficient NOD.Cg-Prkdc scid I l2rg tmlWjl /SzJ (NSG, stock number 005557) mice.
  • mice were purchased from Charles River (Sulzfeld, Germany), Janvier (Le Genest-Saint-lsle, France) or bred within the local animal facility (Zentrale ceremoniesstieranno, Nonethelessstadt, Kunststoff, Germany). All conducted animal experiments were approved by the local regulatory agency (Reg michigan von Oberbayern). Tumor growth was monitored with bioluminescence imaging (BLI) using the In vivo Imaging System Platform Lumina X5 (MS, PerkinElmer, USA) after intraperitoneal (i.p.) injection of substrate (Xenolight D-Luciferin potassium salt, Perkin Elmer, USA) into each mouse according to manufacture's instructions. Afterwards, mice were i.v. treated with PBS or up to 10 7 T cells expressing anti-CD86 CAR, anti-CD33 CAR, anti-CD30 CAR T or control-transduced T cells.
  • mice were treated with either anti-CD86 CAR T cells or anti-CD33 CAR T cells or anti-CD19 CAR T cells as a positive or negative control, respectively. Transduction efficiencies for each experiment were around 40-60%. Furthermore, to prove efficacy of anti- CD86 CAR T cells in NHL models, NHL PDX models were established. To this end, NHL-derived primary patient samples were injected intravenously into NSG mice. Tumor lysis was then either measured by flow cytometry. For flow cytometric measurement of in vivo tumor cells lysis, mice were sacrificed 55 days after tumor cell injection and tumor loads in the spleen, the bone marrow and the blood were quantified by flow cytometry.
  • NHL tumor cells Following antibodies were used to gate for NHL tumor cells: anti-human CD45 (clone 2D1; Biolegend, USA), anti-murine CD45 (clone 30-F11; Biolegend, USA), anti-human CD19 (clone HIB19; Biolegend, USA), antihuman CD3 (clone HIT3a; Biolegend, USA). Dead cells were excluded after staining with a fixable viability dye (eFluorTM 780, eBioscience, USA). Tumor cell counts were quantified using Count BrightTM Absolute Counting Beads (Life Technologies, Darmstadt, Germany).
  • CD86 CAR T cells were tested in AML CDX models. As illustrated in Fig. 19, injection of CD86 CART cells lead to a decrease in luminescence signal. This effect, induced by CD86 CAR T cells, which was not observed in control-treated mice, lead to a sustained tumor remission over a course of 80 days, illustrating the effectiveness of CD86 CAR T cells for treating AML
  • mice were injected with anti-CD86, anti-CD30 or anti-CD19 CAR T cells as a negative control.
  • Treatment with anti-CD86 CAR T cells lead to a complete decrease of bioluminescence signal in all treated mice, comparable to anti- CD30 positive control CAR T cells, while all mice that received anti-CD19 CAR T cells showed an increase in bioluminescence signal (Fig. 20). All mice treated with anti-CD19 CAR T cells eventually succumb to the disease, while 60% of mice treated with CD86 CAR T cells (or anti- CD30 CAR T cells) stayed tumor free (Fig. 20).
  • L-428 HL cells were injected into NSG mice and the mice were treated after tumor onset (i.e., presence of detectable tumor signal, already one day post injection of tumor cells) again with anti-CD86, CD30 or CD19 CAR T cells.
  • Treatment with anti-CD86 or CD30 CAR T cells lead to a diminished bioluminescence signal, whereas the signal in CD19 CAR T cell-treated mice, increased (Fig. 21).
  • anti- CD86 or CD30 CAR T cell-treated mice induced complete tumor remission in all treated mice, while all anti-CD19 CAR T cell treated mice succumb to the disease latest 60 days after tumor injection.
  • the high efficacy of anti-CD86 CAR T cells for treating hematological cancers has been demonstrated.
  • mice were established and used as above described.
  • AML PDX cells were injected into NSG mice and tumor onset in all mice was awaited (Fig. 22). After tumor onset, mice were injected with anti-CD86, anti-CD33 or anti-CD19 CAR T cells. Mice injected with anti-CD86 or anti-CD33 positive control CAR T cells induced complete tumor remission in all treated mice, exemplified by the decrease of luciferase tumor signal in the mice. In contrast, mice treated with anti-CD19 CAR T cells showed an increase in BLI signal, attributable to rapid tumor growth in these mice.
  • PDX NHL derived of a patient suffering from mantle cell lymphoma, MCL
  • BLI could not be used in this model, as PDX samples could not be transduced with fLuc.
  • Successful tumor onset i.e., the presence of detectable tumor signal
  • mice were treated with anti-CD86 CAR T cells, untransduced T cells or PBS. Mice were sacrificed 55 days after tumor injection and efficacy of anti-CD86 CAR T cells were analyzed at this endpoint.
  • Treatment with anti-CD86 CAR T cells lead to significant reduction of the weight of the spleens, compared to mice that were treated with either PBS or untransduced T cells (Fig. 23A). This reduction can be attributed to the reduction of tumor load in the spleen of treated mice as observed in flow cytometric analysis as described in Example 5.3.2., which revealed significant reduction of tumor cells in mice that were treated with anti-CD86 CAR T cells, compared to PBS or untransduced T cells treated mice (Fig. 23B). These results highlight the high effectiveness of anti-CD86 for treating malignant Non-Hodgkin Lymphomas.
  • Tumor cells often orchestrate the formation of a network of immunosuppressive immune cells (Binnewies et al., Nat Med (2016); 24: 541-550). These immunosuppressive cells dampen the anti-tumor responses of the immune system and contribute to the progression of malignant diseases (Nakamura & Smyth, Cellular & Molecular Immunology (2020); 17: 1-12, and Mantovani, EurJ Immunol (2010); 40: 3317-3320).
  • TAM tumor-infiltrating macrophages
  • MDSC myeloid-derived-suppressor cells
  • Tregs T regulatory cells
  • Example 4 the expression of CD86 on immunosuppressive TAMs was analyzed and the efficacy of anti-CD86 CAR T cells to deplete these tumor-suppressive immune cell population was tested. It was thought that, in addition to the direct cytotoxic effect of anti-CD86 CAR T cells, the newly developed therapeutic of the invention would also exhibit additional, indirect, anti-tumorigenic activity.
  • PBMC peripheral blood mononuclear cells
  • MCS magnetic-associated cell sorting
  • macrophages were polarized for 48 hours with 100 ng/ml LPS and 10 ng/ml IFN-y (Mi), 20 ng/ml IL-4 and 20 ng/ml IL-13 (M2), or with cell- free tumor supernatant of L-428 HL cells.
  • macrophages were characterized by flow cytometry using following antibodies: CD80 (clone 2D10; Biolegend, USA), CD86 (clone IT2.2; Biolegend, USA), CD163 (clone GH1/61; Biolegend, USA), CD206 (clone 15-2; Biolegend, USA). Expression of CD86 on different in v/tro-polarized macrophage-trajectories was compared to an isotype control antibody.
  • CDllb clone MI/70; Biolegend, USA
  • CD3 clone OKT3; Biolegend, USA
  • CD69 clone FN50; Biolegend, USA
  • Lysis of macrophages was analyzed with Count BrightTM Absolute Counting Beads (Life Technologies, Darmstadt, Germany) after gating on CDllb + macrophages.
  • T cell activation was measured by measurement of percentages of CD69 + cells after gating on CD3 + T cells.
  • Anti-CD86 CAR T cells efficiently lysed in v/tro-polarized macrophages as observed by reduction of measured CDllb + counts in co-cultures with CD86 CAR T cells and as compared to co-cultures with anti-CD30 or anti-CD19 CAR T cells (Fig. 25A).
  • the anti-CD86 CAR T cells also released high amounts of I FNy into co-culture supernatants (Fig. 25B).
  • the anti-CD86 CAR T cells showed the strongest proliferation in co-cultures with Mh-polarized macrophages compared to Mi and M2-polarized macrophages, illustrated by trace dilution (shift of the FarRed proliferation dye peak to the left, Fig. 25C).
  • Anti-CD30 or anti-CD19 CAR T cells did not proliferate in co-cultures with in v/tro-polarized macrophages of any condition (Fig. 25C).
  • Example 1 the expression of CD86 on various malignancies and the role of anti-CD86 CAR T cells for treating hematological disorders was illustrated.
  • anti-CD86 CAR T cells were at least as effective as established CAR T cells currently undergoing clinical investigation.
  • Example 5 therapeutically relevant situations were tested, in which CD86 CAR T cells exhibited superior functionality relative to established therapies, e.g., at least further exhibiting indirect anti-tumor activity by depleting immunosuppressive cell populations.
  • PDX-388 cells were i.v. injected into NSG mice and disease onset in the mice was awaited. After tumor onset (i.e., as determined by the presence of detectable tumor signal via BLI), a single-dose of anti-CD86 CAR T cells, anti-CD33 or anti-CD19 CAR T cells were injected into PDX-388 tumor-bearing mice.
  • mice that received CD86 CAR T cells rapidly cleared the tumor, illustrated by reduction of BLI signal in these mice (Fig. 26).
  • mice that were treated with anti-CD33 or anti-CD19 CAR T cells showed an increase of the BLI signal (Fig. 26).
  • This treatment response induced by anti-CD86 CAR T cells led to a significantly prolonged overall survival, compared to mice treated with anti-CD33 or anti-CD86 CAR T cells (Fig. 26).
  • L-428-CD30 /_ cells were generated as previously described in Cadilha et al., Science Advances (2021); 7: eabi5781.
  • a two-component guide RNA (gRNA) method was used, and tumor cells were pulsed using a 4D-Nucleofector (Lonza, Switzerland).
  • gRNAs were designed and selected from CHOPCHOPv3 (Labun et al.; Nucleic Acids Res (2019):47:W171-W174) and subsequently synthesized by Integrated DNA technologies (IDT, USA).
  • Successful knockout was verified by flow cytometric staining for CD30.
  • CD30-negative cells were sorted using a BD FACSAriaTM III cell sorter. Polyclonally sorted L-428-CD30 /_ cells were used for all subsequent experiments.
  • L-428-CD30 knockout (L-428-CD30 _/ j cells were generated as described in Example 5.5.2. 3.5 x 10 6 L-428 CD30 cells were injected intravenously into NSG mice. Tumor progression was monitored using BLI. After measurable tumor signal, mice received a single dose of either anti-CD86, anti-CD30 or anti-CD19 CAR T cells. BLI signal in mice treated with anti-CD86 CAR T cells rapidly declined, while mice that received anti-CD30 or anti-CD19 CAR T cells showed increases in BLI signal after treatment (Fig. 27).
  • a total of 266 different specimen of publicly available datasets were used, and the datasets were integrated into a cross-organ-off-tumor-transcriptomic atlas (COOTA).
  • COOTA cross-organ-off-tumor-transcriptomic atlas
  • CD86 was only minimally ( ⁇ 2% of cells) expressed on pre-defined "critical cell clusters" (endothelial cells, alveolar cells, astrocytes, Neurons, oligodendrocytes, cardio myocytes, podocytes, tubule cells, hematopoietic stem cells, Fig. 28).
  • critical cell clusters endothelial cells, alveolar cells, astrocytes, Neurons, oligodendrocytes, cardio myocytes, podocytes, tubule cells, hematopoietic stem cells, Fig. 28.
  • other target antigens from the screening algorithms such as PD-L1
  • single cell analysis was used to compare the expression of CD86 with the known AML- associated target antigens CD33 and CD123 on hematopoietic stem cells.
  • CD86 was found to be significantly lower on hematopoietic stem or hematopoietic progenitor cells (HSC, HPC) when compared to CD33 and CD123 (Fig. 29). These results indicate that CD86 is a more specific target for treating AML than CD33 or CD123 and that anti-CD86 CAR therapy, can spare these cell populations.
  • CB CD34+ stem cells obtained from Stemcell Technologies. All cells were collected after informed consent in accordance with the Declaration of Helsinki. CB CD34+ cells were thawed in a pre-warmed water bath at 37°. Directly after thawing, cells were expanded using StemSpan II Medium (Stemcell Technologies, Vancouver, Canada), supplemented with serum-free nutrient supply and UM729 small molecule inhibitor. For HSC assays and FACS analysis, cells were expanded a total of 7 days, medium was changed after 3 days.
  • Anti-CD86-CAR and anti-CD33-CAR T cells were generated as described in Example 5.2.3.
  • Human CD34+ BM- or CB-derived hematopoietic stem cells were obtained as described in Example 5.6.2.
  • PBMC were isolated from healthy donors using density centrifugation (see Example 5.2.3).
  • CD86 is a more specific and improved marker for AML as compared to CD33
  • the expression of CD86 and CD33 by CD34+ and CD38-negative HSC and by CD34-positive, CD38-positive HPC was determined by FACS.
  • Stem cells were purchased and cultivated as described in Example 5.6.2.
  • FACS analysis was carried out as described in Example 5.1.3.3.
  • FACS antibodies were used for expression analysis of HSCs (Fig. 30): anti-human CD33 (clone WM53; Biolegend, USA), anti-human CD123 (clone 6H6; Biolegend, USA), antihuman CD34 (clone 561; Biolegend, USA), anti-human CD38 (clone HB-7; Biolegend, USA), anti-human CD86 (clone IT2.2, Biolegend, USA).
  • Samples were analyzed using BD LSRFortessaTM II. Dead cells were excluded after staining with a fixable viability dye (eFluorTM 780, eBioscience, USA).
  • FACS antibodies were used for co-culture experiments with CAR T cells and human HSC (Example 5.6.3, Fig. 30): anti-human CD3 (clone HIT3a; Biolegend, USA) antihuman CD33 (clone WM53; Biolegend, USA), anti-human CD34 (clone 561; Biolegend, USA), anti-human CD38 (clone HB-7; Biolegend, USA), anti-human CD45RA (clone HI100, Biolegend, USA), anti-human CD90 (clone 5E10, Biolegend, USA) anti-human CD86 (clone IT2.2; Biolegend, USA). Samples were analyzed using BD LSRFortessaTM II. Dead cells were excluded after staining with a fixable viability dye (eFluorTM 780, eBioscience, USA).
  • a fixable viability dye eFluorTM 780, eBioscience, USA.
  • CD86 was expressed on a smaller subset of cells (48.3%) as compared to CD33 (98.9%).
  • anti-CD33, anti-CD86 CAR T cells or untransduced T cells were mixed with human BM-derived CD34+ cells to a final volume of 200 pl per well in a flat bottom 96 well plate in an effectontarget cell ratio as indicated in the respective Fig. 30B. All cells were cultured in IMDM containing 2% FCS and 0,5% penicillin streptomycin. After 48 hours target cell lysis was determined using FACS (see Example 5.5.3). 5.6.7 FACS analysis of co-cultures
  • Target specificity of anti-CD86 CAR T cells when compared to anti-CD33 CAR T cells was assessed by determining T cell-mediated killing of HSPC and HSC.
  • T cells were isolated and genetically modified to express either anti-CD86-CAR and anti-CD33-CAR as described in Example 5.2.3.
  • HSPC and transduced or untransduced T cells were co-cultured for 48h as described in Example 5.6.3, and T cell-mediated killing was measured by FACS analysis. To quantify the cell numbers Count BrightTM Absolute Counting Beads were used (Example 5.5.2). As shown in Fig. 30B, anti-CD86 CAR T cells show lower killing of HSPC when compared to anti- CD33 CAR T cells.
  • Standard hybridoma-based techniques were used to develop and identify further exemplary antibodies that bind the extracellular region of CD86.
  • peptide immunization and hybridoma development were performed as known in the art (see, e.g. Parray et al., Int Immunopharmacol. (2020); 85:106639).
  • Recombinant CD86 protein or the immunization peptide were immobilized on a plate. Following blocking and washing of the plate as known in the art, hybridoma supernatants were added, allowed to incubate, and, standard ELISA detection methods used for detection of binders, e.g. light emission. Optical density was determined using standard methodologies. No antigen conditions were used as negative controls. Results of the tested clones are provided in Figure 31.

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Abstract

La présente invention concerne la reconnaissance de CD86 en tant que marqueur du cancer hématologique et concerne ainsi des agents de ciblage de CD86 pour le traitement de tels cancers, en particulier, la leucémie aiguë myéloïde (LAM), le lymphome de Hodgkin (LH) et le lymphome non hodgkinien (LNH). L'invention concerne en particulier un lymphocyte exprimant par recombinaison un récepteur de lymphocyte T antigénique chimérique (CAR) comprenant un domaine de liaison à l'antigène qui se lie spécifiquement à CD86 pour une utilisation dans le traitement de tels cancers. L'invention concerne également la construction de CAR, c'est-à-dire comprenant un domaine de liaison à l'antigène qui se lie spécifiquement à CD86.
EP24702779.0A 2023-01-30 2024-01-30 Lymphocytes exprimant des car anti-cd86 pour thérapie tumorale ciblée Pending EP4658298A1 (fr)

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EP23154047 2023-01-30
PCT/EP2024/052269 WO2024160845A1 (fr) 2023-01-30 2024-01-30 Lymphocytes exprimant des car anti-cd86 pour thérapie tumorale ciblée

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US5858358A (en) 1992-04-07 1999-01-12 The United States Of America As Represented By The Secretary Of The Navy Methods for selectively stimulating proliferation of T cells
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US5585362A (en) 1989-08-22 1996-12-17 The Regents Of The University Of Michigan Adenovirus vectors for gene therapy
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US7067318B2 (en) 1995-06-07 2006-06-27 The Regents Of The University Of Michigan Methods for transfecting T cells
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ES2836743T3 (es) 2014-06-02 2021-06-28 Us Health Receptores de antígeno quiméricos que seleccionan como diana CD-19
MX2018004503A (es) 2015-10-15 2019-04-15 Us Health Receptores de antigeno quimerico anti-cd30.

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WO2024160845A1 (fr) 2024-08-08

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