WO2023102328A2

WO2023102328A2 - Treatment of cd30-positive cancer

Info

Publication number: WO2023102328A2
Application number: PCT/US2022/080232
Authority: WO
Inventors: Cliona M. Rooney; David H. Quach
Original assignee: Baylor College of Medicine
Current assignee: Baylor College of Medicine
Priority date: 2021-12-03
Filing date: 2022-11-21
Publication date: 2023-06-08
Anticipated expiration: 2024-06-03
Also published as: TW202339777A; WO2023102328A3

Abstract

Embodiments of the present disclosure provide methods of treating a CD30-positive cancer in a subject. In specific embodiments, an effective dose of CD30-specific chimeric antigen receptor (CAR)-expressing T cells are administered to the subject, wherein the dose may be administered in two parts at two time points, wherein a first part of the dose is administered at a first time point and the remaining part of the dose is administered at a second time point, wherein the first and second time points are 2 to 4 days apart.

Description

Treatment of CD30-positive cancer

This application claims priority to U.S. Provisional Patent Application Serial No. 63/285,539, filed December 3, 2021 , which is incorporated by reference herein in its entirety.

Field of the Invention

The present invention relates to methods of medical treatment.

Background

CD30.CAR-T therapy comprises T-cells genetically modified to express a chimeric antigen receptor (CAR) specific for CD30, to target and kill cancer cells expressing the CD30 transmembrane glycoprotein. The drug product is generated from peripheral blood mononuclear cells (PBMCs) taken from patients with CD30-positive lymphoma.

In an initial Phase 1 study in patients with CD30-positive hematologic malignancies including classical Hodgkin Lymphoma (cHL), autologous CD30.CAR-T administration in the absence of lymphodepleting chemotherapy was proved to be safe but only a minority of patients had durable responses (NCT01316146; Ramos et al., J Clin Invest. (2017) 127(9):3462-3471 ).

CD30.CAR-T therapy has been shown to be well-tolerated, with significant clinical activity demonstrated in heavily pre-treated patients with CD30-positive, relapsed or refractory classical HL and some NHL patients, following lymphodepletion chemotherapy (NCT02917083 (RELY-30); Ramos et al., Biol Blood Marrow Transplant 25 (2019) S7-S75, Abstract 79).

Summary of the Invention

In a first aspect, the present disclosure provides a method of treating a CD30-positive cancer in a subject, comprising administering a dose of CD30-specific chimeric antigen receptor (CAR)-expressing T cells to the subject, wherein the dose is administered in two parts at two time points, wherein a first part of the dose is administered at a first time point and the remaining part of the dose is administered at a second time point, wherein the first and second time points are 2 to 4 days apart.

In a second aspect, the present disclosure provides a composition comprising CD30-specific chimeric antigen receptor (CAR)-expressing T cells for use in a method of treating a CD30-positive cancer, wherein the method comprises administering a dose of CD30-specific chimeric antigen receptor (CAR)- expressing T cells to the subject, wherein the dose is administered in two parts at two time points, wherein a first part of the dose is administered at a first time point and the remaining part of the dose is administered at a second time point, wherein the first and second time points are 2 to 4 days apart. In a third aspect, the present disclosure provides a use of a composition comprising CD30-specific chimeric antigen receptor (CAR)-expressing T cells in the manufacture of a medicament for use in a method of treating a CD30-positive cancer, wherein the method comprises administering a dose of CD30- specific chimeric antigen receptor (CAR)-expressing T cells to the subject, wherein the dose is administered in two parts at two time points, wherein a first part of the dose is administered at a first time point and the remaining part of the dose is administered at a second time point, wherein the first and second time points are 2 to 4 days apart.

In some embodiments, the CD30-specific chimeric antigen receptor (CAR)-expressing T cells are allogeneic to the subject.

In a fourth aspect, the present disclosure provides a method of eliminating alloreactive T cells in a subject with a CD30-positive cancer, comprising administering a dose of allogeneic CD30-specific chimeric antigen receptor (CAR)-expressing T cells to the subject, wherein a first part of the dose is administered at a first time point and the remaining part of the dose is administered at a second time point, wherein the first and second time points are 2 to 4 days apart.

In a fifth aspect, the present disclosure provides a composition comprising allogeneic CD30-specific chimeric antigen receptor (CAR)-expressing T cells for use in a method of eliminating alloreactive T cells in a subject with a CD30-positive cancer, wherein the method comprises administering a dose of CD30- specific chimeric antigen receptor (CAR)-expressing T cells to the subject, wherein the dose is administered in two parts at two time points, wherein a first part of the dose is administered at a first time point and the remaining part of the dose is administered at a second time point, wherein the first and second time points are 2 to 4 days apart.

In a sixth aspect, the present disclosure provides a use of a composition comprising allogeneic CD30- specific chimeric antigen receptor (CAR)-expressing T cells in the manufacture of a medicament for use in a method of eliminating alloreactive T cells in a subject with a CD30-positive cancer, wherein the method comprises administering a dose of CD30-specific chimeric antigen receptor (CAR)-expressing T cells to the subject, wherein the dose is administered in two parts at two time points, wherein a first part of the dose is administered at a first time point and the remaining part of the dose is administered at a second time point, wherein the first and second time points are 2 to 4 days apart.

In some embodiments, the method comprises adoptive transfer of allogeneic CD30-specific chimeric antigen receptor (CAR)-expressing T cells.

In some embodiments, the CD30-specific chimeric antigen receptor (CAR)-expressing T cells are virusspecific T cells.

In some embodiments, the virus-specific T cells are specific for Epstein-Barr virus (EBV).

In some embodiments, the first and second time points are 3 days apart.

In some embodiments, 50% of the dose is administered at the first time point, and 50% of the dose is administered at the second time point. In some embodiments, 50% of the dose is administered on day 0, and 50% of the dose is administered on day 3.

In some embodiments, the dose is about 4 x 10⁷ to about 4 x 10⁸ CD30-specific CAR-expressing T cells/m² to the subject.

In some embodiments, the dose is about 4 x 10⁷ CD30-specific CAR-expressing T cells/m².

In some embodiments, the dose is about 1 x 10⁸ CD30-specific CAR-expressing T cells/m².

In some embodiments, the dose is about 4 x 10⁸ CD30-specific CAR-expressing T cells/m².

In a seventh aspect, the present disclosure provides a method of treating a CD30-positive cancer in a subject, comprising administering CD30-specific chimeric antigen receptor (CAR)-expressing T cells to the subject, wherein the method comprises administering a first dose of the CD30-specific chimeric antigen receptor (CAR)-expressing T cells to the subject, and subsequently administering a second dose of the CD30-specific chimeric antigen receptor (CAR)-expressing T cells to the subject, wherein the first and second doses are administered 2 to 4 days apart.

In an eighth aspect, the present disclosure provides a composition comprising CD30-specific chimeric antigen receptor (CAR)-expressing T cells for use in a method of treating a CD30-positive cancer, wherein the method comprises administering a first dose of the CD30-specific chimeric antigen receptor (CAR)-expressing T cells to the subject, and subsequently administering a second dose of the CD30- specific chimeric antigen receptor (CAR)-expressing T cells to the subject, wherein the first and second doses are administered 2 to 4 days apart.

In a ninth aspect, the present disclosure provides a use of a composition comprising CD30-specific chimeric antigen receptor (CAR)-expressing T cells in the manufacture of a medicament for use in a method of treating a CD30-positive cancer, wherein the method comprises administering a first dose of the CD30-specific chimeric antigen receptor (CAR)-expressing T cells to the subject, and subsequently administering a second dose of the CD30-specific chimeric antigen receptor (CAR)-expressing T cells to the subject, wherein the first and second doses are administered 2 to 4 days apart.

In some embodiments, the virus-specific T cells are specific for Epstein-Barr virus.

In some embodiments, the first and second doses are administered 3 days apart.

In some embodiments, the first dose is administered on day 0 and the second dose is administered on day 3. In some embodiments, the first dose is about 2 x 10⁷ to about 2 x 10⁸ CD30-specific CAR-expressing T cells/m².

In some embodiments, the second dose is about 2 x 10⁷ to about 2 x 10⁸ CD30-specific CAR-expressing T cells/m².

In some embodiments, the total dose comprising the first and second dose is about 4 x 10⁷ to about 4 x 10⁸ CD30-specific CAR-expressing T cells/m².

In some embodiments, the total dose comprising the first and second dose is about 4 x 10⁷ CD30-specific CAR-expressing T cells/m².

In some embodiments, the total dose comprising the first and second dose is about 1 x 10⁸ CD30-specific CAR-expressing T cells/m².

In some embodiments, the total dose comprising the first and second dose is about 4 x 10⁸ CD30-specific CAR-expressing T cells/m².

In a tenth aspect, the present disclosure provides a method of treating a CD30-positive cancer in a subject, comprising administering a CD30-specific chimeric antigen receptor (CAR)-expressing T cells to the subject, wherein the method comprises administering a total dose of about 4 x 10⁷ to about 4 x 10⁸ CD30-specific CAR-expressing T cells/m² to the subject, wherein the total dose comprises a first dose and a second dose, wherein the first and second dose are administered 2 to 4 days apart.

In an eleventh aspect, the present disclosure provides a composition comprising CD30-specific chimeric antigen receptor (CAR)-expressing T cells for use in a method of treating a CD30-positive cancer in a subject, wherein the method comprises administering CD30-specific chimeric antigen receptor (CAR)- expressing T cells to the subject, wherein the method comprises administering a total dose of about 4 x 10⁷ to about 4 x 10⁸ CD30-specific CAR-expressing T cells/m² to the subject, wherein the total dose comprises a first dose and a second dose, wherein the first and second dose are administered 2 to 4 days apart.

In a twelfth aspect, the present disclosure provides a use of a composition comprising CD30-specific chimeric antigen receptor (CAR)-expressing T cells in the manufacture of a medicament for use in a method of treating a CD30-positive cancer in a subject, wherein the method comprises administering CD30-specific chimeric antigen receptor (CAR)-expressing T cells to the subject, wherein the method comprises administering a total dose of about 4 x 10⁷ to about 4 x 10⁸ CD30-specific CAR-expressing T cells/m² to the subject, wherein the total dose comprises a first dose and a second dose, wherein the first and second dose are administered 2 to 4 days apart.

In some embodiments, the method comprises adoptive transfer of allogeneic CD30-specific chimeric antigen receptor (CAR)-expressing T cells. In some embodiments, the CD30-specific chimeric antigen receptor (CAR)-expressing T cells are virusspecific T cells.

In some embodiments, the first and second dose are administered 3 days apart.

In some embodiments, the first dose is 50% of the total dose, and the second dose is 50% of the total dose.

In some embodiments, the first and second doses are about 2 x 10⁷ to about 2 x 10⁸ CD30-specific CAR- expressing T cells/m².

In some embodiments, the total dose is about 4 x 10⁷ to about 4 x 10⁸ CD30-specific CAR-expressing T cells/m².

In some embodiments, the total dose is about 4 x 10⁷ CD30-specific CAR-expressing T cells/m².

In some embodiments, the total dose is about 1 x 10⁸ CD30-specific CAR-expressing T cells/m².

In some embodiments, the total dose is about 4 x 10⁸ CD30-specific CAR-expressing T cells/m².

In some embodiments, prior to administration of the CD30-specific chimeric antigen receptor (CAR)- expressing T cells, a lymphodepleting chemotherapy is administered to the subject.

In some embodiments, the lymphodepleting chemotherapy comprises fludarabine and cyclophosphamide.

In some embodiments, fludarabine is administered at a dose of 15 to 60 mg/m² per day, for 2 to 6 consecutive days.

In some embodiments, fludarabine is administered at a dose of 30 mg/m² per day, for 3 consecutive days.

In some embodiments, cyclophosphamide is administered at a dose of 250 to 1000 mg/m² per day, for 2 to 6 consecutive days.

In some embodiments, cyclophosphamide is administered at a dose of 500 mg/m² per day, for 3 consecutive days.

In some embodiments, fludarabine is administered at a dose of 30 mg/m² per day and cyclophosphamide is administered at a dose of 500 mg/m² per day to a subject for 3 consecutive days.

In some embodiments, the lymphodepleting chemotherapy comprises cyclophosphamide and bendamustine.

In some embodiments, cyclophosphamide is administered at a dose of 500 mg/m² per day, for 3 consecutive days. In some embodiments, bendamustine is administered at a dose of 35 to 140 mg/m² per day, for 2 to 6 consecutive days.

In some embodiments, bendamustine is administered at a dose of 70 mg/m² per day, for 3 consecutive days.

In some embodiments, cyclophosphamide is administered at a dose of 500 mg/m² per day and bendamustine is administered at a dose of 70 mg/m² per day to a subject for 3 consecutive days.

In some embodiments, the CD30-positive cancer is selected from: a hematological cancer, a solid cancer, a hematopoietic malignancy, Hodgkin’s lymphoma, anaplastic large cell lymphoma, peripheral T cell lymphoma, peripheral T cell lymphoma not otherwise specified, T cell leukemia, T cell lymphoma, cutaneous T cell lymphoma, NK-T cell lymphoma, extranodal NK-T cell lymphoma, non-Hodgkin’s lymphoma, B cell non-Hodgkin’s lymphoma, diffuse large B cell lymphoma, diffuse large B cell lymphoma not otherwise specified, EBV-positive B cell lymphoma, EBV-positive diffuse large B cell lymphoma, primary mediastinal B cell lymphoma, advanced systemic mastocytosis, a germ cell tumor and testicular embryonal carcinoma.

In some embodiments, the CD30-positive cancer is selected from: Hodgkin’s lymphoma, non-Hodgkin’s lymphoma, anaplastic large cell lymphoma, peripheral T cell lymphoma not otherwise specified, extranodal NK-T cell lymphoma, diffuse large B cell lymphoma not otherwise specified and primary mediastinal large B-cell lymphoma.

In some embodiments, the subject has previously failed therapy for the CD30-positive cancer.

In some embodiments, the CD30-positive cancer is a relapsed or refractory CD30-positive cancer.

In some embodiments, the CD30-specific CAR-expressing T cells comprise a CAR comprising: (i) an antigen-binding domain which binds specifically to CD30, (ii) a transmembrane domain, and (iii) a signalling domain, wherein the signalling domain comprises: (a) an amino acid sequence derived from the intracellular domain of CD28, and (b) an amino acid sequence comprising an immunoreceptor tyrosinebased activation motif (ITAM).

In some embodiments, the signalling domain comprises an amino acid sequence having at least 80% amino acid sequence identity to SEQ ID NO:26.

In some embodiments, the transmembrane domain is derived from the transmembrane domain of CD28.

In some embodiments, the transmembrane domain comprises an amino acid sequence having at least 80% amino acid sequence identity to SEQ ID NO:20.

In some embodiments, the antigen-binding domain comprises an amino acid sequence having at least 80% amino acid sequence identity to SEQ ID NO:14, and an amino acid sequence having at least 80% amino acid sequence identity to SEQ ID NO:15.

In some embodiments, the antigen-binding domain comprises an amino acid sequence having at least 80% amino acid sequence identity to SEQ ID NO:18. In some embodiments, the signalling domain comprises: (a) an amino acid sequence derived from the intracellular domain of CD3£.

In some embodiments, the signalling domain comprises an amino acid sequence having at least 80% amino acid sequence identity to SEQ ID NO:25.

In some embodiments, the CAR additionally comprises a hinge region provided between the antigenbinding domain and the transmembrane domain.

In some embodiments, the hinge region comprises an amino acid sequence having at least 80% amino acid sequence identity to SEQ ID NO:33.

In some embodiments, the CAR comprises an amino acid sequence having at least 80% amino acid sequence identity to SEQ ID NO:35 or 36.

The invention includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided.

Summary of the Figures

Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures in which:

Figure 1. Schematic of method for assessing CD30 expression in T cells. Alloreactive host T cells from donors A, B and C were generated by 1 , 2 or 3 stimulations with allogeneic irradiated polymorphonuclear cells from HLA-mismatched donors. To evaluate if repeated encounters of allogeneic mismatched graft PBMCs affect CD30 expression on host T cells, host T cells that remained after the second or third boost were subsequently co-cultured with graft PBMCs from the same donor used for priming. CD30 expression on alloreactive host T cells was evaluated at 24, 48 and 72 hours of co-culture. (n=3 donor pairs).

Figure 2. (A) Host T cells that were not primed were co-cultured with their respective mismatched graft PBMCs. Low levels CD30 was observed to be expressed in CD4 and CD8 T cells. (B) Alloreactive host T cells that underwent 2 rounds of stimulation were co-cultured with their respective mismatched graft PBMCs. CD30 expression was observed to be up-regulated in both host CD4 and CD8 T cell compared to their un-primed counterparts. (C) Alloreactive host T cells that underwent 3 rounds of stimulation were co-cultured with their respective mismatched graft PBMCs. CD30 expression was observed to be highly expressed in both host CD4 and CD8 T cells compared to their un-primed counterparts. Experiment was performed with 3 donor pairs.

Figure 3. CD30 expression on T-cells activated with antibodies to CD3 and CD28. Non-tissues culture treated 24 well plates were coated with CD3 and CD28 antibodies overnight. Coated wells were washed with PBS and PBMCs were cultured at 1 .0 x 10⁶ cells per well in RPMI 1640 medium supplemented with 10% FBS and 1% GlutaMAX. Cells were harvested for CD30 analysis on the days indicated. N=3. Figure 4. CD30 expression on alloreactive cells. CD4+ and CD8+ T-cells were sorted from PBMC, then 1 .0 x 105 CD4+ or CD8+ T-cells were co-cultured with either 5.0 x 10³ irradiated allogeneic lymphoblastoid cell lines (LCL), 5.0 x 10³ irradiated HLA-negative ULCL or were left unstimulated. Cells were cultured in 96-well round bottom plates in RPMI 1640 medium supplemented with 10% FBS and 1% GlutaMAX. Cells were harvested for CD30 analysis on the days indicated. N=3.

Detailed Description of the Invention

Aspects and embodiments of the present invention will now be discussed with reference to the accompanying figures. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.

CD30-positive cancer

The present disclosure relates to the treatment of cancer, more particularly CD30-positive cancer.

CD30 (also known as TNFRSF8) is the protein identified by UniProt: P28908. CD30 is a single pass, type I transmembrane glycoprotein of the tumor necrosis factor receptor superfamily. CD30 structure and function is described e.g. in van der Weyden et al., Blood Cancer Journal (2017) 7: e603 and Muta and Podack Immunol. Res. (2013) 57(1 -3) :151 -8, both of which are hereby incorporated by reference in their entirety.

Alternative splicing of mRNA encoded by the human TNFRSF8 gene yields three isoforms: isoform 1 (‘long’ isoform; UniProt: P28908-1 , v1 ; SEQ ID NO:1 ), isoform 2 (‘cytoplasmic’, ‘short’ or ‘C30V’ isoform, UniProt: P28908-2; SEQ ID NO:2) in which the amino acid sequence corresponding to positions 1 to 463 of SEQ ID NOU are missing, and isoform 3 (UniProt: P28908-3; SEQ ID NO:3) in which the amino acid sequence corresponding to positions 1 to 111 and position 446 of SEQ ID NOU are missing. The N- terminal 18 amino acids of SEQ ID NOU form a signal peptide (SEQ ID NO:4), which is followed by a 367 amino acid extracellular domain (positions 19 to 385 of SEQ ID NOU , shown in SEQ ID NO:5), a 21 amino acid transmembrane domain (positions 386 to 406 of SEQ ID NOU , shown in SEQ ID NO:6), and a 189 amino acid cytoplasmic domain (positions 407 to 595 of SEQ ID NOU , shown in SEQ ID NOT).

In this specification “CD30” refers to CD30 from any species and includes CD30 isoforms, fragments, variants or homologues from any species. As used herein, a “fragment”, “variant” or “homologue” of a reference protein may optionally be characterised as having at least 60%, preferably one of 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of the reference protein (e.g. a reference isoform). In some embodiments fragments, variants, isoforms and homologues of a reference protein may be characterised by ability to perform a function performed by the reference protein.

In some embodiments, the CD30 is from a mammal e.g. a primate (rhesus, cynomolgous, or human) and/or a rodent (e.g. rat or murine) CD30. In preferred embodiments the CD30 is a human CD30. Isoforms, fragments, variants or homologues may optionally be characterised as having at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of an immature or mature CD30 isoform from a given species, e.g. human. A fragment of CD30 may have a minimum length of one of 10, 20, 30, 40, 50, 100, 200, 300, 400, 500 or 590 amino acids, and may have a maximum length of one of 10, 20, 30, 40, 50, 100, 200, 300, 400, 500 or 595 amino acids.

In some embodiments, the CD30 comprises, or consists of, an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:1 , 2 or 3.

In some embodiments, the CD30 comprises, or consists of, an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:5.

In some embodiments, a fragment of CD30 comprises, or consists of, an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:5 or 19.

The present disclosure relates to the treatment of CD30-associated cancer.

As used herein, “cancer” may refer to any unwanted cell proliferation (or any disease manifesting itself by unwanted cell proliferation), neoplasm or tumor. The cancer may be benign or malignant and may be primary or secondary (metastatic). A neoplasm or tumor may be any abnormal growth or proliferation of cells and may be located in any tissue. The cancer may be of tissues/cells derived from e.g. the adrenal gland, adrenal medulla, anus, appendix, bladder, blood, bone, bone marrow, brain, breast, cecum, central nervous system (including or excluding the brain) cerebellum, cervix, colon, duodenum, endometrium, epithelial cells (e.g. renal epithelia), gallbladder, oesophagus, glial cells, heart, ileum, jejunum, kidney, lacrimal glad, larynx, liver, lung, lymph, lymph node, lymphoblast, maxilla, mediastinum, mesentery, myometrium, nasopharynx, omentum, oral cavity, ovary, pancreas, parotid gland, peripheral nervous system, peritoneum, pleura, prostate, salivary gland, sigmoid colon, skin, small intestine, soft tissues, spleen, stomach, testis, thymus, thyroid gland, tongue, tonsil, trachea, uterus, vulva, and/or white blood cells.

In some embodiments the cancer is a cancer in which CD30 is pathologically implicated. That is, in some embodiments the cancer is a cancer which is caused or exacerbated by CD30 expression, a cancer for which expression of CD30 is a risk factor and/or a cancer for which expression of CD30 is positively associated with onset, development, progression, severity or metastasis of the cancer. The cancer may be characterised by CD30 expression, e.g. the cancer may comprise cells expressing CD30. Such cancers may be referred to as CD30-positive cancers.

A CD30-positive cancer may be a cancer comprising cells expressing CD30 (e.g. cells expressing CD30 protein at the cell surface). A CD30-positive cancer may overexpress CD30. Overexpression of CD30 can be determined by detection of a level of gene or protein expression of CD30 which is greater than the level of expression by equivalent non-cancerous cells/non-tumor tissue. A given cancer/sample may be evaluated for gene/protein expression of CD30 by techniques well known to the skilled person, e.g. by qRT-PCR (for gene expression), antibody-based assays (e.g. western blot, flow cytometry, etc. for protein expression).

CD30-positive cancers are described e.g. in van der Weyden et al., Blood Cancer Journal (2017) 7:e603 and Muta and Podack, Immunol Res (2013), 57(1 -3):151 -8, both of which are hereby incorporated by reference in their entirety. CD30 is also expressed on activated T and B lymphocytes, and by various lymphoid neoplasms including classical Hodgkin’s lymphoma and anaplastic large cell lymphoma. Variable expression of CD30 has also been shown for peripheral T cell lymphoma, not otherwise specified (PTCL-NOS), adult T cell leukemia/lymphoma, cutaneous T cell lymphoma (CTCL), extra-nodal NK-T cell lymphoma, various B cell non-Hodgkin’s lymphomas (including diffuse large B cell lymphoma, particularly EBV-positive diffuse large B cell lymphoma), and advanced systemic mastocytosis. CD30 expression has also been observed in some non-hematopoietic malignancies, including germ cell tumors and testicular embryonal carcinomas.

The transmembrane glycoprotein CD30, is a member of the tumor necrosis factor receptor superfamily (Falini et al., Blood (1995) 85(1 ):1 -14). Members of the TNF/TNF-receptor (TNF-R) superfamily coordinate the immune response at multiple levels and CD30 plays a role in regulating the function or proliferation of normal lymphoid cells. CD30 was originally described as an antigen recognized by a monoclonal antibody, Ki-1 , which was raised by immunizing mice with a HL-derived cell line, L428 (Muta and Podack, Immunol Res (2013) 57: 151 -158). CD30 antigen expression has been used to identify ALCL and Reed-Sternberg cells in Hodgkin's disease (Falini et al., Blood (1995) 85(1 ):1 -14). With the wide expression in the lymphoma malignant cells, CD30 is therefore a potential target for developing both antibody-based immunotherapy and cellular therapies. Importantly, CD30 is not typically expressed on normal tissues under physiologic conditions, thus is notably absent on resting mature or precursor B or T cells (Younes and Ansell, Semin Hematol (2016) 53: 186-189). Brentuximab vedotin, an antibody-drug conjugate that targets CD30 was initially approved for the treatment of CD30-positive HL (Adcetris® US Package Insert 2018). Data from brentuximab vedotin trials support CD30 as a therapeutic target for the treatment of CD30-positive lymphoma, although toxicities associated with its use are of concern.

Hodgkin lymphoma (HL) is an uncommon malignancy involving lymph nodes and the lymphatic system. The incidence of HL by age is bimodal with most patients diagnosed between 15 and 30 years of age, followed by another peak in adults aged 55 years or older. In 2019 it is estimated there will be 8,110 new cases (3,540 in females and 4570 in males) in the United States and 1 ,000 deaths (410 female and 590 males) from this disease (American Cancer Society 2019). Based on 2012-2016 cases in National Cancer Institute’s SEER database, the incidence rate for HL for the pediatric HL patients in US is as follows: Age 1 -4: 0.1 ; Age 5-9: 0.3; Age 10-14: 1.3; Age 15-19: 3.3 per 100,000 (SEER Cancer Statistics Review, 1975-2016]).

The World Health Organization (WHO) classification divides HL into 2 main types: classical Hodgkin lymphoma (cHL) and nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL). In Western countries, cHL accounts for 95% and NLPHL accounts for 5% of all HL (National Comprehensive Cancer Network Guidelines 2019).

First-line chemotherapy for cHL patients with advanced disease is associated with cure rates between 70% and 75% (Karantanos et al., Blood Lymphat Cancer (2017) 7:37-52). Salvage chemotherapy followed by Autologous Stem Cell Transplant (ASCT) is commonly used in patients who relapse after primary therapy. Unfortunately, up to 50% of the cHL patients experience disease recurrence after ASCT. The median overall survival of patients who relapse after ASCT is approximately two years (Alinari Blood (2016) 127:287-295). Despite aggressive combination chemotherapy, between 10% and 40% of patients do not achieve a response to salvage chemotherapy and there are no randomized clinical trial data supporting ASCT in non-responders. For patients who do not respond to salvage chemotherapy, relapse after ASCT or who are not candidates for this approach, the prognosis continues to be grave and new treatment approaches are urgently needed (Keudell British Journal of Haematology (2019) 184:105-1 12).

While a majority of the pediatric population (children, adolescents, and young adults) will be cured with currently available therapy, a small fraction of patients may have refractory or relapsed disease and require novel therapies that have an acceptable safety profile with improved efficacy benefit (Flerlage et al., Blood (2018) 132: 376-384; Kelly, Blood (2015) 126: 2452-2458; McClain and Kamdar, in UpToDate 2019; Moskowitz, ASCO Educational Book (2019) 477-486). HL patients treated with high dose chemotherapy during childhood commonly experience treatment-related long-term sequelae, such as cardiac, pulmonary, gonadal, and endocrine toxicity as well as second malignant neoplasms (Castellino et al., Blood (201 1 ) 1 17(6): 1806-1816).

In some embodiments, a CD30-positive cancer according to the present disclosure may be selected from: a hematological cancer, a solid cancer, a hematopoietic malignancy, Hodgkin’s lymphoma, anaplastic large cell lymphoma, peripheral T cell lymphoma, peripheral T cell lymphoma not otherwise specified, T cell leukemia, T cell lymphoma, cutaneous T cell lymphoma, NK-T cell lymphoma, extranodal NK-T cell lymphoma, non-Hodgkin’s lymphoma, B cell non-Hodgkin’s lymphoma, diffuse large B cell lymphoma, diffuse large B cell lymphoma not otherwise specified, EBV-positive B cell lymphoma, EBV-positive diffuse large B cell lymphoma, primary mediastinal B cell lymphoma, advanced systemic mastocytosis, a germ cell tumor and testicular embryonal carcinoma.

The CD30-positive cancer may be a relapsed CD30-positive cancer. As used herein, a “relapsed” cancer refers to a cancer which responded to a treatment (e.g. a first line therapy for the cancer), but which has subsequently re-emerged/progressed, e.g. after a period of remission. For example, a relapsed cancer may be a cancer whose growth/progression was inhibited by a treatment (e.g. a first line therapy for the cancer), and which has subsequently grown/progressed.

The CD30-positive cancer may be a refractory CD30-positive cancer. As used herein, a “refractory” cancer refers to a cancer which has not responded to a treatment (e.g. a first line therapy for the cancer). For example, a refractory cancer may be a cancer whose growth/progression was not inhibited by a treatment (e.g. a first line therapy for the cancer). In some embodiments a refractory cancer may be a cancer for which a subject receiving treatment for the cancer did not display a partial or complete response to the treatment.

In embodiments where the CD30-positive cancer is anaplastic large cell lymphoma, the cancer may be relapsed or refractory with respect to treatment with chemotherapy, brentuximab vedotin, or crizotinib.

In embodiments where the CD30-positive cancer is peripheral T cell lymphoma not otherwise specified, the cancer may be relapsed or refractory with respect to treatment with chemotherapy or brentuximab vedotin.

In embodiments where the CD30-positive cancer is extranodal NK-T cell lymphoma, the cancer may be relapsed or refractory with respect to treatment with chemotherapy (with or without asparaginase) or brentuximab vedotin.

In embodiments where the CD30-positive cancer is diffuse large B cell lymphoma not otherwise specified, the cancer may be relapsed or refractory with respect to treatment with chemotherapy (with or without rituximab) or CD19 CAR-T therapy.

In embodiments where the CD30-positive cancer is primary mediastinal B cell lymphoma, the cancer may be relapsed or refractory with respect to treatment with chemotherapy, immune checkpoint inhibitor (e.g. PD-1 inhibitor) or CD19 CAR-T therapy.

The CD30-specific chimeric antigen receptor (CAR)-expressing T cells of the present disclosure, for example the CD30.CAR-EBVSTs, are also useful in the treatment of EBV-positive (EBV+) lymphoma/cancer, or EBV-associated lymphomas/cancers. The EBV+ lymphoma/cancer or EBV- associated lymphoma/cancer may be CD30-positive. The EBV+ lymphoma/cancer or EBV-associated lymphoma/cancer may be CD30-negative.

Thus, the methods, compositions, and use of compositions disclosed herein may also be used to treat EBV+ lymphoma/cancer or EBV-associated lymphoma/cancer. In some embodiments, the EBV+ lymphoma/cancer or EBV-associated lymphoma/cancer is CD30-positive. In other embodiments, the EBV+ lymphoma/cancer or EBV-associated lymphoma/cancer is CD30-negative. In some embodiments, the EBV+ lymphoma/cancer is EBV-positive B cell lymphoma or EBV-positive diffuse large cell B cell lymphoma.

In some embodiments, the cancer is selected from the group consisting of: a CD30-positive cancer, an EBV-associated cancer, a hematological cancer, a myeloid hematologic malignancy, a hematopoietic malignancy, a lymphoblastic hematologic malignancy, myelodysplastic syndrome, leukemia, T cell leukemia, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, lymphoma, Hodgkin’s lymphoma, non-Hodgkin’s lymphoma, B cell non-Hodgkin’s lymphoma, diffuse large B cell lymphoma, primary mediastinal B cell lymphoma, EBV-associated lymphoma, EBV-positive B cell lymphoma, EBV-positive diffuse large B cell lymphoma, EBV-positive lymphoma associated with X-linked lymphoproliferative disorder, EBV-positive lymphoma associated with HIV infection/AIDS, oral hairy leukoplakia, Burkitt’s lymphoma, post-transplant lymphoproliferative disease, central nervous system lymphoma, anaplastic large cell lymphoma, T cell lymphoma, ALK-positive anaplastic T cell lymphoma, ALK-negative anaplastic T cell lymphoma, peripheral T cell lymphoma, cutaneous T cell lymphoma, NK-T cell lymphoma, extra-nodal NK-T cell lymphoma, thymoma, multiple myeloma, a solid cancer, epithelial cell cancer, gastric cancer, gastric carcinoma, gastric adenocarcinoma, gastrointestinal adenocarcinoma, liver cancer, hepatocellular carcinoma, cholangiocarcinoma, head and neck cancer, head and neck squamous cell carcinoma, oral cavity cancer, oropharyngeal cancer, oropharyngeal carcinoma, oral cancer, laryngeal cancer, nasopharyngeal carcinoma, oesophageal cancer, colorectal cancer, colorectal carcinoma, colon cancer, colon carcinoma, cervical carcinoma, prostate cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, squamous lung cell carcinoma, bladder cancer, urothelial carcinoma, skin cancer, melanoma, advanced melanoma, renal cell cancer, renal cell carcinoma, ovarian cancer, ovarian carcinoma, mesothelioma, breast cancer, brain cancer, glioblastoma, prostate cancer, pancreatic cancer, mastocytosis, advanced systemic mastocytosis, germ cell tumor or testicular embryonal carcinoma.

CD30-specific CARs

CARs

The present disclosure relates to immune cells comprising/expressing CD30-specific chimeric antigen receptors (CARs).

In some embodiments, the CD30-specific chimeric antigen receptor (CAR) is expressed as a transgene on immune cells.

Chimeric Antigen Receptors (CARs) are recombinant receptor molecules which provide both antigenbinding and T cell activating functions. CAR structure and engineering is reviewed, for example, in Dotti et al., Immunol Rev (2014) 257(1 ), which is hereby incorporated by reference in its entirety.

CARs comprise an antigen-binding domain linked via a transmembrane domain to a signalling domain. An optional hinge or spacer domain may provide separation between the antigen-binding domain and transmembrane domain, and may act as a flexible linker. When expressed by a cell, the antigen-binding domain is provided in the extracellular space, and the signalling domain is intracellular.

The antigen-binding domain mediates binding to the target antigen for which the CAR is specific. The antigen-binding domain of a CAR may be based on the antigen-binding region of an antibody which is specific for the antigen to which the CAR is targeted. For example, the antigen-binding domain of a CAR may comprise amino acid sequences for the complementarity-determining regions (CDRs) of an antibody which binds specifically to the target antigen. The antigen-binding domain of a CAR may comprise or consist of the light chain and heavy chain variable region amino acid sequences of an antibody which binds specifically to the target antigen. The antigen-binding domain may be provided as a single chain variable fragment (scFv) comprising the sequences of the light chain and heavy chain variable region amino acid sequences of an antibody. Antigen-binding domains of CARs may target antigen based on other protein protein interaction, such as ligand :receptor binding; for example an IL-13Ra2-targeted CAR has been developed using an antigen-binding domain based on IL-13 (see e.g. Kahlon et al. 2004 Cancer Res 64(24): 9160-9166).

The transmembrane domain is provided between the antigen-binding domain and the signalling domain of the CAR. The transmembrane domain provides for anchoring the CAR to the cell membrane of a cell expressing a CAR, with the antigen-binding domain in the extracellular space, and signalling domain inside the cell. Transmembrane domains of CARs may be derived from transmembrane region sequences for cell membrane-bound proteins (e.g. CD28, CD8, etc.).

Throughout this specification, polypeptides, domains and amino acid sequences which are ‘derived from’ a reference polypeptide/domain/amino acid sequence have at least 60%, preferably one of 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of the reference polypeptide/domain/amino acid sequence. Polypeptides, domains and amino acid sequences which are ‘derived from’ a reference polypeptide/domain/amino acid sequence preferably retains the functional and/or structural properties of the reference polypeptide/domain/amino acid sequence.

By way of illustration, an amino acid sequence derived from the intracellular domain of CD28 may comprise an amino acid sequence having 60%, preferably one of 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the intracellular domain of CD28, e.g. as shown in SEQ ID NO:26. Furthermore, an amino acid sequence derived from the intracellular domain of CD28 preferably retains the functional properties of the amino acid sequence of SEQ ID NO:26, i.e. the ability activate CD28-mediated signalling.

The amino acid sequence of a given polypeptide or domain thereof can be retrieved from, or determined from a nucleic acid sequence retrieved from, databases known to the person skilled in the art. Such databases include GenBank, EMBL and UniProt.

The signalling domain comprises amino acid sequences required activation of immune cell function. The CAR signalling domains may comprise the amino acid sequence of the intracellular domain of CD3-£, which provides immunoreceptor tyrosine-based activation motifs (ITAMs) for phosphorylation and activation of the CAR-expressing cell. Signalling domains comprising sequences of other ITAM-containing proteins have also been employed in CARs, such as domains comprising the ITAM containing region of FcyRI (Haynes et al., 2001 J Immunol 166(1 ):182-187). CARs comprising a signalling domain derived from the intracellular domain of CD3-£ are often referred to as first generation CARs.

The signalling domains of CARs typically also comprise the signalling domain of a costimulatory protein (e.g. CD28, 4-1 BB etc.), for providing the costimulation signal necessary for enhancing immune cell activation and effector function. CARs having a signalling domain including additional co-stimulatory sequences are often referred to as second generation CARs. In some cases CARs are engineered to provide for co-stimulation of different intracellular signalling pathways. For example, CD28 costimulation preferentially activates the phosphatidylinositol 3-kinase (P13K) pathway, whereas 4-1 BB costimulation triggers signalling is through TNF receptor associated factor (TRAF) adaptor proteins. Signalling domains of CARs therefore sometimes contain co-stimulatory sequences derived from signalling domains of more than one co-stimulatory molecule. CARs comprising a signalling domain with multiple co-stimulatory sequences are often referred to as third generation CARs.

An optional hinge or spacer region may provide separation between the antigen-binding domain and the transmembrane domain, and may act as a flexible linker. Such regions may be or comprise flexible domains allowing the binding moiety to orient in different directions, which may e.g. be derived from the CH1 -CH2 hinge region of IgG.

Through engineering to express a CAR specific for a particular target antigen, immune cells (typically T cells, but also other immune cells such as NK cells) can be directed to kill cells expressing the target antigen. Binding of a CAR-expressing T cell (CAR-T cell) to the target antigen for which it is specific triggers intracellular signalling, and consequently activation of the T cell. The activated CAR-T cell is stimulated to divide and produce factors resulting in killing of the cell expressing the target antigen.

CD30-specific CARs

Since cHL is apparently sensitive to the cellular immune response (graft versus lymphoma effect) and antibody treatment, there is interest in combining both approaches through the generation of artificial chimeric antigen receptors (CARs).

CAR-targeting CD30 in preclinical studies have shown that T-lymphocytes engineered to express this receptor are redirected to kill CD30-positive HL cell lines (Hornbach et al. Cancer Res. (1998) 58(6) : 1 1 16- 9, Savoldo et al. Blood (2007) 1 10(7):2620-30). Further to this, in vitro and in vivo experiments to examine potential on-target toxicity, showed that anti-CD30 CAR-T cells demonstrated specific cytotoxicity against CD30-positive lymphoma cells while sparing CD30-positive activated HSPCs and B lymphocytes (Hornbach et al., Mol Ther (2016) 24: 1423-1434).

An in vitro assessment of CD30.CAR T Cells that were manufactured as part of an ongoing clinical study was conducted (NCT01316146; Ramos et al., J Clin Invest. (2017) 127(9):3462-3471 ; NCT02917083; Ramos et al., Biol Blood Marrow Transplant 25 (2019) S7-S75, Abstract 79). The starting material for the engineered T cells was peripheral blood mononuclear cells from lymphoma patients. The manufactured CD30.CAR T cells in this published study were transduced with the same retroviral vector as the final drug product. A total of 22 lots of CD30.CAR T Cells were manufactured using either IL-2 (1 1 products) or IL-7/IL-15 (1 1 products). By day 15 of culture, CD30.CAR T Cells grown in IL-7/IL-15 had greater expansion from baseline and higher final cell numbers (45 ± 13 and 1.2 x 109 ± 5.5 x 108, respectively) than those expanded in IL-2 (27.4 ± 13 and 6.5 x 108 ± 3.3 x 108, respectively). CAR expression was comparable in both groups (>89%).

Specific in vitro cytotoxicity of the CD30.CAR T Cells was demonstrated in a 4-hour 51 Cr release assay, using effector to target ratios of 40:1 , 20:1 , 10:1 , and 5:1 . The HDLM-2 cell line was used as a CD30- positive target cell while CD30-negative Raji tumor cells were used as a control (Ctr-Ts). A total of n=9 lots of cells cultured in IL-2 were tested, while a total of n=8 lots of cells expanded in IL-7/IL-15 were tested. Figure 2D of Ramos et al., J Clin Invest. (2017) 127(9):3462-3471 shows mean specific lysis, provides evidence of the proposed mechanism of action of CD30.CAR-T, as shown by direct, specific, cellular cytotoxicity against CD30-positive tumor cells.

Antigen-binding domain

An “antigen-binding domain” refers to a domain which is capable of binding to a target antigen. The target antigen of the CARs of the present disclosure is CD30, or fragment thereof. Antigen-binding domains according to the present disclosure may be derived from an antibody/antibody fragment (e.g. Fv, scFv, Fab, single chain Fab (scFab), single domain antibodies (e.g. VhH), etc.) directed against CD30, or another CD30-binding molecule (e.g. a target antigen-binding peptide or nucleic acid aptamer, ligand or other molecule).

In some embodiments, the antigen-binding domain comprises an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) of an antibody capable of specific binding to the CD30. In some embodiments, the domain capable of binding to a target antigen comprises or consists of a CD30-binding peptide/polypeptide, e.g. a peptide aptamer, thioredoxin, monobody, anticalin, Kunitz domain, avimer, knottin, fynomer, atrimer, DARPin, affibody, nanobody (i.e. a single-domain antibody (sdAb)) affilin, armadillo repeat protein (ArmRP), OBody or fibronectin - reviewed e.g. in Reverdatto et al., Curr Top Med Chem. 2015; 15(12): 1082-1101 , which is hereby incorporated by reference in its entirety (see also e.g. Boersma et al., J Biol Chem (2011 ) 286:41273-85 and Emanuel et al., Mabs (2011 ) 3:38-48).

The antigen-binding domains of the present disclosure may be derived from the VH and a VL of an antibody capable of specific binding to CD30. Antibodies generally comprise six complementaritydetermining regions CDRs; three in the heavy chain variable region (VH): HC-CDR1 , HC-CDR2 and HC- CDR3, and three in the light chain variable region (VL): LC-CDR1 , LC-CDR2, and LC-CDR3. The six CDRs together define the paratope of the antibody, which is the part of the antibody which binds to the target antigen. The VH region and VL region comprise framework regions (FRs) either side of each CDR, which provide a scaffold for the CDRs. From N-terminus to C-terminus, VHs comprise the following structure: N term-[HC-FR1]-[HC-CDR1]-[HC-FR2]-[HC-CDR2]-[HC-FR3]-[HC-CDR3]-[HC-FR4]-C term; and VLs comprise the following structure: N term-[LC-FR1]-[LC-CDR1]-[LC-FR2]-[LC-CDR2]-[LC-FR3]- [LC-CDR3]-[LC-FR4]-C term. VH and VL sequences may be provided in any suitable format provided that the antigen-binding domain can be linked to the other domains of the CAR. Formats contemplated in connection with the antigenbinding domain of the present disclosure include those described in Carter, Nat. Rev. Immunol 2006, 6: 343-357, such as scFv, dsFV, (scFv)2 diabody, triabody, tetrabody, Fab, minibody, and F(ab)2 formats.

In some embodiments, the antigen-binding domain comprises the CDRs of an antibody/antibody fragment which is capable of binding to CD30. In some embodiments, the antigen-binding domain comprises the VH region and the VL region of an antibody/antibody fragment which is capable of binding to CD30. A moiety comprised of the VH and a VL of an antibody may also be referred to herein as a variable fragment (Fv). The VH and VL may be provided on the same polypeptide chain, and joined via a linker sequence; such moieties are referred to as single-chain variable fragments (scFvs). Suitable linker sequences for the preparation of scFv are known to the skilled person, and may comprise serine and glycine residues.

In some embodiments, the antigen-binding domain comprises, or consists of, Fv capable of binding to CD30. In some embodiments, the antigen-binding domain comprises, or consists of, a scFv capable of binding to CD30.

In some embodiments, the CD30 binding domain is derived from CD30 ligand.

The CD30-binding domain of the CAR of the present disclosure preferably displays specific binding to CD30 or a fragment thereof. The CD30-binding domain of the CAR of the present disclosure preferably displays specific binding to the extracellular domain of CD30. The CD30-binding domain may be derived from an anti-CD30 antibody or other CD30-binding agent, e.g. a CD30-binding peptide or CD30-binding small molecule.

The CD30-binding domain may be derived from the antigen-binding moiety of an anti-CD30 antibody.

Anti-CD30 antibodies include HRS3 and HRS4 (described e.g. in Hornbach et al., Scand J Immunol (1998) 48(5):497-501 ), HRS3 derivatives described in Schlapschy et al., Protein Engineering, Design and Selection (2004) 17(12): 847-860, BerH2 (MBL International Cat# K0145-3, RRID:AB_590975), SGN-30 (also known as cAC10, described e.g. in Forero-Torres et al., Br J Haematol (2009) 146:171-9), MDX- 060 (described e.g. in Ansell et al., J Clin Oncol (2007) 25:2764-9; also known as 5F11 , iratumumab), and MDX-1401 (described e.g. in Cardarelli et al., Clin Cancer Res. (2009) 15(10):3376-83), and anti- CD30 antibodies described in WO 2020/068764 A1 , WO 2003/059282 A2, WO 2006/089232 A2, WO 2007/084672 A2, WO 2007/044616 A2, WO 2005/001038 A2, US 2007/166309 A1 , US 2007/258987 A1 , WO 2004/010957 A2 and US 2005/009769 A1 .

In some embodiments a CD30-binding domain according to the present disclosure comprises the CDRs of an anti-CD30 antibody. In some embodiments a CD30-binding domain according to the present disclosure comprises the VH and VL regions of an anti-CD30 antibody. In some embodiments a CD30- binding domain according to the present disclosure comprises an scFv comprising the VH and VL regions of an anti-CD30 antibody.

There are several different conventions for defining antibody CDRs and FRs, such as those described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991 ), Chothia et al., J. Mol. Biol. 196:901 -917 (1987), and VBASE2, as described in Retter et al., Nucl. Acids Res. (2005) 33 (suppl 1 ): D671 -D674. The CDRs and FRs of the VH regions and VL regions of the antibodies described herein are defined according to VBASE2.

In some embodiments the antigen-binding domain of the present disclosure comprises: a VH incorporating the following CDRs:

HC-CDR1 having the amino acid sequence of SEQ ID NO:8

HC-CDR2 having the amino acid sequence of SEQ ID NO:9

HC-CDR3 having the amino acid sequence of SEQ ID NQ:10, or a variant thereof in which one or two or three amino acids in one or more of HC-CDR1 , HC-CDR2, or HC-CDR3 are substituted with another amino acid; and a VL incorporating the following CDRs:

LC-CDR1 having the amino acid sequence of SEQ ID NO:11

LC-CDR2 having the amino acid sequence of SEQ ID NO:12

LC-CDR3 having the amino acid sequence of SEQ ID NO:13, or a variant thereof in which one or two or three amino acids in one or more of LC-CDR1 , LC-CDR2, or LC-CDR3 are substituted with another amino acid.

In some embodiments the antigen-binding domain comprises: a VH comprising, or consisting of, an amino acid sequence having at least 80% sequence identity (e.g. at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) to the amino acid sequence of SEQ ID NO:14; and a VL comprising, or consisting of, an amino acid sequence having at least 80% sequence identity (e.g. at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) to the amino acid sequence of SEQ ID NO:15.

In some embodiments, a CD30-binding domain may comprise or consist of a single chain variable fragment (scFv) comprising a VH sequence and a VL sequence as described herein. The VH sequence and VL sequence may be covalently linked. In some embodiments, the VH and the VL sequences are linked by a flexible linker sequence, e.g. a flexible linker sequence as described herein. The flexible linker sequence may be joined to ends of the VH sequence and VL sequence, thereby linking the VH and VL sequences. In some embodiments the VH and VL are joined via a linker sequence comprising, or consisting of, the amino acid sequence of SEQ ID NO:16 or 17. In some embodiments, the CD30-binding domain comprises, or consists of, an amino acid sequence having at least 80%, 85% 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:18.

In some embodiments the CD30-binding domain is capable of binding to CD30, e.g. in the extracellular domain of CD30. In some embodiments, the CD30-binding domain is capable of binding to the epitope of CD30 which is bound by antibody HRS3, e.g. within the region of amino acid positions 185-335 of human CD30 numbered according to SEQ ID NO:1 , shown in SEQ ID NO:19 (Schlapschy et al., Protein Engineering, Design and Selection (2004) 17(12): 847-860, hereby incorporated by reference in its entirety).

In some embodiments, a CD30-binding domain may comprise or consist of a single chain variable fragment (scFv) comprising a VH sequence and a VL sequence as described herein. The VH sequence and VL sequence may be covalently linked. In some embodiments, the VH and the VL sequences are linked by a flexible linker sequence, e.g. a flexible linker sequence as described herein. The flexible linker sequence may be joined to ends of the VH sequence and VL sequence, thereby linking the VH and VL sequences. In some embodiments the VH and VL are joined via a linker sequence comprising, or consisting of, the amino acid sequence of SEQ ID NO:16.

In some embodiments, the antigen-binding domain (and thus the CAR) is multispecific. By “multispecific” it is meant that the antigen-binding domain displays specific binding to more than one target. In some embodiments the antigen-binding domain is a bispecific antigen-binding domain. In some embodiments the antigen-binding molecule comprises at least two different antigen-binding moieties (i.e. at least two antigen-binding moieties, e.g. comprising non-identical VHs and VLs). Individual antigen-binding moieties of multispecific antigen-binding domains may be connected, e.g. via linker sequences.

The antigen-binding domain may bind to at least two, non-identical target antigens, and so is at least bispecific. The term “bispecific” means that the antigen-binding domain is able to bind specifically to at least two distinct antigenic determinants. At least one of the target antigens for the multispecific antigenbinding domain/CAR may be CD30.

It will be appreciated that an antigen-binding domain according to the present disclosure (e.g. a multispecific antigen-binding domain) comprises antigen-binding moieties capable of binding to the target(s) for which the antigen-binding domain is specific. For example, an antigen-binding domain which is capable of binding to CD30 and an antigen other than CD30 may comprise: (i) an antigen-binding moiety which is capable of binding to CD30, and (ii) an antigen-binding moiety which is capable of binding to a target antigen other than CD30. Transmembrane domain

The CAR of the present disclosure comprises a transmembrane domain. A transmembrane domain refers to any three-dimensional structure formed by a sequence of amino acids which is thermodynamically stable in a biological membrane, e.g. a cell membrane. In connection with the present disclosure, the transmembrane domain may be an amino acid sequence which spans the cell membrane of a cell expressing the CAR.

The transmembrane domain may comprise or consist of a sequence of amino acids which forms a hydrophobic alpha helix or beta-barrel. The amino acid sequence of the transmembrane domain of the CAR of the present disclosure may be, or may be derived from, the amino acid sequence of a transmembrane domain of a protein comprising a transmembrane domain. Transmembrane domains are recorded in databases such as GenBank, UniProt, Swiss-Prot, TrEMBL, Protein Information Resource, Protein Data Bank, Ensembl, and InterPro, and/or can be identified/predicted e.g. using amino acid sequence analysis tools such as TMHMM (Krogh et al., 2001 J Mol Biol 305: 567-580).

In some embodiments, the amino acid sequence of the transmembrane domain of the CAR of the present disclosure may be, or may be derived from, the amino acid sequence of the transmembrane domain of a protein expressed at the cell surface. In some embodiments the protein expressed at the cell surface is a receptor or ligand, e.g. an immune receptor or ligand. In some embodiments the amino acid sequence of the transmembrane domain may be, or may be derived from, the amino acid sequence of the transmembrane domain of one of ICOS, ICOSL, CD86, CTLA-4, CD28, CD80, MHC class I a, MHC class II a, MHC class II p, CD3e, CD36, CD3y, CD3- , TCRa TCRp, CD4, CD8a, CD8p, CD40, CD40L, PD-1 , PD-L1 , PD-L2, 4-1 BB, 4-1 BBL, 0X40, OX40L, GITR, GITRL, TIM-3, Galectin 9, LAG3, CD27, CD70, LIGHT, HVEM, TIM-4, TIM-1 , ICAM1 , LFA-1 , LFA-3, CD2, BTLA, CD160, LILRB4, LILRB2, VTCN1 , CD2, CD48, 2B4, SLAM, CD30, CD30L, DR3, TL1 A, CD226, CD155, CD112 and CD276. In some embodiments, the transmembrane is, or is derived from, the amino acid sequence of the transmembrane domain of CD28, CD3- , CD8a, CD8p or CD4. In some embodiments, the transmembrane is, or is derived from, the amino acid sequence of the transmembrane domain of CD28.

In some embodiments, the transmembrane domain comprises, or consists of, an amino acid sequence having at least 80%, 85% 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:20.

In some embodiments, the transmembrane domain comprises, or consists of, an amino acid sequence having at least 80%, 85% 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:21 .

In some embodiments, the transmembrane domain comprises, or consists of, an amino acid sequence having at least 80%, 85% 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:22. Signalling domain

The chimeric antigen receptor of the present disclosure comprises a signalling domain. The signalling domain provides sequences for initiating intracellular signalling in cells expressing the CAR.

The signalling domain comprises ITAM-containing sequences. An ITAM-containing sequence comprises one or more immunoreceptor tyrosine-based activation motifs (ITAMs). ITAMs comprise the amino acid sequence YXXL/I (SEQ ID NO:23), wherein “X” denotes any amino acid. In ITAM-containing proteins, sequences according to SEQ ID NO:23 are often separated by 6 to 8 amino acids; YXXL/I (X)6-8YXXL/I (SEQ ID NO:24). When phosphate groups are added to the tyrosine residue of an ITAM by tyrosine kinases, a signalling cascade is initiated within the cell.

In some embodiments, the signalling domain comprises one or more copies of an amino acid sequence according to SEQ ID NO:23 or SEQ ID NO:24. In some embodiments, the signalling domain comprises at least 1 , 2, 3, 4, 5 or 6 copies of an amino acid sequence according to SEQ ID NO:23. In some embodiments, the signalling domain comprises at least 1 , 2, or 3 copies of an amino acid sequence according to SEQ ID NO:24.

In some embodiments, the signalling domain comprises an amino acid sequence which is, or which is derived from, the amino acid sequence of an ITAM-containing sequence of a protein having an ITAM- containing amino acid sequence. In some embodiments the signalling domain comprises an amino acid sequence which is, or which is derived from, the amino acid sequence of the intracellular domain of one of CD3- , FcyRI, CD3e, CD36, CD3y, CD79a, CD79p, FcyRIIA, FcyRIIC, FcyRIIIA, FcyRIV or DAP12. In some embodiments the signalling domain comprises an amino acid sequence which is, or which is derived from, the intracellular domain of CD3- .

In some embodiments, the signalling domain comprises an amino acid sequence which comprises, or consists of, an amino acid sequence having at least 80%, 85% 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:25.

The signalling domain may additionally comprise one or more costimulatory sequences. A costimulatory sequence is an amino acid sequence which provides for costimulation of the cell expressing the CAR of the present disclosure. Costimulation promotes proliferation and survival of a CAR-expressing cell upon binding to the target antigen, and may also promote cytokine production, differentiation, cytotoxic function and memory formation by the CAR-expressing cell. Molecular mechanisms of T cell costimulation are reviewed in Chen and Flies, 2013 Nat Rev Immunol 13(4):227-242.

A costimulatory sequence may be, or may be derived from, the amino acid sequence of a costimulatory protein. In some embodiments the costimulatory sequence is an amino acid sequence which is, or which is derived from, the amino acid sequence of the intracellular domain of a costimulatory protein. Upon binding of the CAR to the target antigen, the costimulatory sequence provides costimulation to the cell expressing the CAR costimulation of the kind which would be provided by the costimulatory protein from which the costimulatory sequence is derived upon ligation by its cognate ligand. By way of example in the case of a CAR comprising a signalling domain comprising a costimulatory sequence derived from CD28, binding to the target antigen triggers signalling in the cell expressing the CAR of the kind that would be triggered by binding of CD80 and/or CD86 to CD28. Thus a costimulatory sequence is capable of delivering the costimulation signal of the costimulatory protein from which the costimulatory sequence is derived.

In some embodiments, the costimulatory protein may be a member of the B7-CD28 superfamily (e.g. CD28, ICOS), or a member of the TNF receptor superfamily (e.g. 4-1 BB, 0X40, CD27, DR3, GITR, CD30, HVEM). In some embodiments, the costimulatory sequence is, or is derived from, the intracellular domain of one of CD28, 4-1 BB, ICOS, CD27, 0X40, HVEM, CD2, SLAM, TIM-1 , CD30, GITR, DR3, CD226 and LIGHT. In some embodiments, the costimulatory sequence is, or is derived from, the intracellular domain of CD28.

In some embodiments the signalling domain comprises more than one non-overlapping costimulatory sequences. In some embodiments the signalling domain comprises 1 , 2, 3, 4, 5 or 6 costimulatory sequences. Plural costimulatory sequences may be provided in tandem.

Whether a given amino acid sequence is capable of initiating signalling mediated by a given costimulatory protein can be investigated e.g. by analysing a correlate of signalling mediated by the costimulatory protein (e.g. expression/activity of a factor whose expression/activity is upregulated or downregulated as a consequence of signalling mediated by the costimulatory protein).

Costimulatory proteins upregulate expression of genes promoting cell growth, effector function and survival through several transduction pathways. For example, CD28 and ICOS signal through phosphatidylinositol 3 kinase (PI3K) and AKT to upregulate expression of genes promoting cell growth, effector function and survival through NF-KB, mTOR, NFAT and AP1/2. CD28 also activates AP1/2 via CDC42/RAC1 and ERK1/2 via RAS, and ICOS activates C-MAF. 4-1 BB, 0X40, and CD27 recruit TNF receptor associated factor (TRAF) and signal through MAPK pathways, as well as through PI3K.

In some embodiments the signalling domain comprises a costimulatory sequence which is, or which is derived from CD28.

In some embodiments, the signalling domain comprises a costimulatory sequence which comprises, or consists of, an amino acid sequence having at least 80%, 85% 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:26. Kofler et al. Mol. Ther. (2011 ) 19: 760-767 describes a variant CD28 intracellular domain in which the lek kinase binding site is mutated in order to reduce induction of IL-2 production on CAR ligation, in order to minimise regulatory T cell-mediated suppression of CAR-T cell activity. The amino acid sequence of the variant CD28 intracellular domain is shown in SEQ ID NO:27.

In some embodiments, the signalling domain comprises a costimulatory sequence which comprises, or consists of, an amino acid sequence having at least 80%, 85% 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:27.

In some embodiments, the signalling domain comprises, or consists of, an amino acid sequence having at least 80%, 85% 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:28.

Hinge region

The CAR may further comprise a hinge region. The hinge region may be provided between the antigenbinding domain and the transmembrane domain. The hinge region may also be referred to as a spacer region. A hinge region is an amino acid sequence which provides for flexible linkage of the antigenbinding and transmembrane domains of the CAR.

The presence, absence and length of hinge regions has been shown to influence CAR function (reviewed e.g. in Dotti et al., Immunol Rev (2014) 257(1 ) supra).

In some embodiments, the CAR comprises a hinge region which comprises, or consists of, an amino acid sequence which is, or which is derived from, the CH1-CH2 hinge region of human IgG 1 , a hinge region derived from CD8a, e.g. as described in WO 2012/031744 A1 , or a hinge region derived from CD28, e.g. as described in WO 2011/041093 A1 . In some embodiments, the CAR comprises a hinge region derived from the CH1 -CH2 hinge region of human IgG 1 .

In some embodiments, the hinge region comprises, or consists of, an amino acid sequence having at least 80%, 85% 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:29 or 30.

In some embodiments, the CAR comprises a hinge region which comprises, or consists of, an amino acid sequence which is, or which is derived from, the CH2-CH3 region (i.e. the Fc region) of human IgG 1 .

In some embodiments, the hinge region comprises, or consists of, an amino acid sequence having at least 80%, 85% 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:31 . Hornbach et al., Gene Therapy (2010) 17:1206-1213 describes a variant CH2-CH3 region for reduced activation of FcyR-expressing cells such as monocytes and NK cells. The amino acid sequence of the variant CH2-CH3 region is shown in SEQ ID NO:32.

In some embodiments, the hinge region comprises, or consists of, an amino acid sequence having at least 80%, 85% 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:32.

In some embodiments, the hinge region comprises, or consists of: an amino acid sequence which is, or which is derived from, the CH1 -CH2 hinge region of human IgG 1 , and an amino acid sequence which is, or which is derived from, the CH2-CH3 region (i.e. the Fc region) of human IgG 1 .

In some embodiments, the hinge region comprises, or consists of, an amino acid sequence having at least 80%, 85% 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:33.

Additional sequences

The CAR may additionally comprise a signal peptide (also known as a leader sequence or signal sequence). Signal peptides normally consist of a sequence of 5-30 hydrophobic amino acids, which form a single alpha helix. Secreted proteins and proteins expressed at the cell surface often comprise signal peptides. Signal peptides are known for many proteins, and are recorded in databases such as GenBank, UniProt and Ensembl, and/or can be identified/predicted e.g. using amino acid sequence analysis tools such as SignalP (Petersen et al., 2011 Nature Methods 8: 785-786) or Signal-BLAST (Frank and Sippl, 2008 Bioinformatics 24: 2172-2176).

The signal peptide may be present at the N-terminus of the CAR, and may be present in the newly synthesised CAR. The signal peptide provides for efficient trafficking of the CAR to the cell surface. Signal peptides are removed by cleavage, and thus are not comprised in the mature CAR expressed by the cell surface.

In some embodiments, the signal peptide comprises, or consists of, an amino acid sequence having at least 80%, 85% 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:34.

In some embodiments the CAR comprises one or more linker sequences between the different domains (i.e. the antigen-binding domain, hinge region, transmembrane domain, signalling domain). In some embodiments the CAR comprises one or more linker sequences between subsequences of the domains (e.g. between VH and VL of an antigen-binding domain).

Linker sequences are known to the skilled person, and are described, for example in Chen et al., Adv Drug Deliv Rev (2013) 65(10): 1357-1369, which is hereby incorporated by reference in its entirety. In some embodiments, a linker sequence may be a flexible linker sequence. Flexible linker sequences allow for relative movement of the amino acid sequences which are linked by the linker sequence. Flexible linkers are known to the skilled person, and several are identified in Chen et al., Adv Drug Deliv Rev (2013) 65(10): 1357-1369. Flexible linker sequences often comprise high proportions of glycine and/or serine residues. In some embodiments, the linker sequence comprises at least one glycine residue and/or at least one serine residue. In some embodiments the linker sequence consists of glycine and serine residues. In some embodiments, the linker sequence has a length of 1 -2, 1 -3, 1 -4, 1 -5, 1 -10, 1 -20, 1 -30, 1 -40 or 1 -50 amino acids.

In some embodiments a linker sequence comprises, or consists, of the amino acid sequence shown in SEQ ID NO:16. In some embodiments a linker sequence comprises, or consists, of 1 , 2, 3, 4 or 5 tandem copies of the amino acid sequence shown in SEQ ID NO:16.

The CARs may additionally comprise further amino acids or sequences of amino acids. For example, the antigen-binding molecules and polypeptides may comprise amino acid sequence(s) to facilitate expression, folding, trafficking, processing, purification or detection. For example, the CAR may comprise a sequence encoding a His, (e.g. 6XHis), Myc, GST, MBP, FLAG, HA, E, or Biotin tag, optionally at the N- or C- terminus. In some embodiments the CAR comprises a detectable moiety, e.g. a fluorescent, lunminescent, immuno-detectable, radio, chemical, nucleic acid or enzymatic label.

Particular exemplary CARs

In some embodiments of the present disclosure, the CAR comprises, or consists of: an extracellular moiety of the anti-CD30 HRS3 scFv domain, connected to spacer and hinge domains derived from the CH2-CH3 of human IgG 1 , the transmembrane and intracellular domains of CD28, and the and the intracellular domain of CD3£.

In some embodiments of the present disclosure, the CAR comprises, or consists of:

An antigen-binding domain comprising or consisting of an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 80%, 85% 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:18;

A hinge region comprising or consisting of an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 80%, 85% 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:33;

A transmembrane domain comprising or consisting of an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 80%, 85% 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NQ:20; and

A signalling domain comprising or consisting of an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 80%, 85% 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:28. In some embodiments of the present disclosure, the CAR comprises, or consists of an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 80%, 85% 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:35 or 36.

In some embodiments, the CAR is selected from an embodiment of a CD30-specific CAR described in Hornbach et al. Cancer Res. (1998) 58(6) : 1 1 16-9, Hornbach et al. Gene Therapy (2000) 7:1067-1075, Hornbach et al. J Immunother. (1999) 22(6):473-80, Hornbach et al. Cancer Res. (2001 ) 61 :1976-1982, Hornbach et al. J Immunol (2001 ) 167:6123-6131 , Savoldo et al. Blood (2007) 1 10(7):2620-30, Koehler et al. Cancer Res. (2007) 67(5):2265-2273, Di Stasi et al. Blood (2009) 1 13(25):6392-402, Hornbach et al. Gene Therapy (2010) 17:1206-1213, Chmielewski et al. Gene Therapy (201 1 ) 18:62-72, Kofler et al. Mol. Ther. (201 1 ) 19(4):760-767, Gilham, Abken and Pule. Trends in Mol. Med. (2012) 18(7):377-384, Chmielewski et al. Gene Therapy (2013) 20:177-186, Hornbach et al. Mol. Ther. (2016) 24(8):1423-1434, Ramos et al. J. Clin. Invest. (2017) 127(9) :3462-3471 , WO 2015/028444 A1 or WO 2016/008973 A1 , all of which are hereby incorporated by reference in their entirety.

CD30-specific CAR-expressing T cells

Aspects of the present disclosure relate to immune cells comprising/expressing CD30-specific chimeric antigen receptors (CARs), particularly, CD30-specific CAR-expressing T cells.

It will be appreciated that where cells are referred to herein in the singular (i.e. “a/the cell”), pluralities/populations of such cells are also contemplated.

CAR-expressing T cells may express or comprise a CAR according to the present disclosure. CAR- expressing T cells may comprise or express nucleic acid encoding a CAR according to the present disclosure. It will be appreciated that a CAR-expressing cell comprises the CAR it expresses. It will also be appreciated that a cell expressing nucleic acid encoding a CAR also expresses and comprises the CAR encoded by the nucleic acid.

The T cell may express e.g. CD3 polypeptides (e.g. CD3y CD3e CD3£ or CD36), TCR polypeptides (TCRa or TCRp), CD27, CD28, CD4 or CD8. In some embodiments, the T cell is a CD3+ T cell. In some embodiments, the T cell is a CD3+, CD4+ T cell. In some embodiments, the T cell is a CD3+, CD8+ T cell. In some embodiments, the T cell is a T helper cell (TH cell)). In some embodiments, the T cell is a cytotoxic T cell (e.g. a cytotoxic T lymphocyte (CTL)).

Producing CAR-expressing, virus-specific immune cells

Methods for producing CAR-expressing T cells are well known to the skilled person. They generally involve modifying T cells to express/comprise a CAR, e.g. introducing nucleic acid encoding a CAR into T cells. T cells (may be modified to comprise/express a CAR or nucleic acid encoding a CAR described herein according to methods that are well known to the skilled person. The methods generally comprise nucleic acid transfer for permanent (stable) or transient expression of the transferred nucleic acid.

Any suitable genetic engineering platform may be used to modify a cell according to the present disclosure. Suitable methods for modifying a cell include the use of genetic engineering platforms such as gammaretroviral vectors, lentiviral vectors, adenovirus vectors, DNA transfection, transposon-based gene delivery and RNA transfection, for example as described in Maus et al., Annu Rev Immunol (2014) 32:189-225, hereby incorporated by reference in its entirety.

Methods also include those described e.g. in Wang and Riviere Mol Ther Oncolytics. (2016) 3:16015, which is hereby incorporated by reference in its entirety. Suitable methods for introducing nucleic acid(s)/vector(s) into cells include transduction, transfection and electroporation.

Methods for generating/expanding populations of CAR-expressing T cells in vitro/ex vivo are well known to the skilled person. Suitable culture conditions (i.e. cell culture media, additives, stimulations, temperature, gaseous atmosphere), cell numbers, culture periods and methods for introducing nucleic acid encoding a CAR into cells, etc. can be determined by reference e.g. to Hornbach et al. J Immunol (2001 ) 167:6123-6131 , Ramos et al. J. Clin. Invest. (2017) 127(9):3462-3471 and WO 2015/028444 A1 , all of which are hereby incorporated by reference in their entirety.

Conveniently, cultures of cells according to the present disclosure may be maintained at 37°C in a humidified atmosphere containing 5% CO2. The cells of cell cultures can be established and/or maintained at any suitable density, as can readily be determined by the skilled person.

Cultures can be performed in any vessel suitable for the volume of the culture, e.g. in wells of a cell culture plate, cell culture flasks, a bioreactor, etc. In some embodiments cells are cultured in a bioreactor, e.g. a bioreactor described in Somerville and Dudley, Oncoimmunology (2012) 1 (8):1435-1437, which is hereby incorporated by reference in its entirety. In some embodiments cells are cultured in a GRex cell culture vessel, e.g. a GRex flask or a GRex 100 bioreactor.

The methods may comprise culturing populations of immune cells (e.g. heterogeneous populations of immune cells, e.g. peripheral blood mononuclear cells; PBMCs) comprising cells having antigen-specific receptors (TCRs) in the presence of antigen-presenting cells (APCs) presenting viral antigen peptide:MHC complexes, under conditions providing appropriate costimulation and signal amplification so as to cause activation and expansion. The APCs may be infected with virus encoding, or may comprise/express, the viral antigen/peptide(s), and present the viral antigen peptide in the context of an MHC molecule. Stimulation causes T cell activation, and promotes cell division (proliferation), resulting in generation and/or expansion of a population of T cells specific for the viral antigen. The process of T cell activation is well known to the skilled person and described in detail, for example, in Immunobiology, 5th Edn. Janeway CA Jr, Travers P, Walport M, et al. New York: Garland Science (2001 ), Chapter 8, which is incorporated by reference in its entirety.

The population of cells obtained following stimulation is enriched for T cells specific for the virus as compared to the population prior to stimulation (i.e. the virus-specific T cells are present at an increased frequency in the population following stimulation). In this way, a population of T cells specific for the virus is expanded/generated out of a heterogeneous population of T cells having different specificities. A population of T cells specific for a virus may be generated from a single T cell by stimulation and consequent cell division. An existing population of T cells specific for a virus may be expanded by stimulation and consequent cell division of cells of the population of virus-specific T cells.

T cells may be activated prior to introduction of nucleic acid encoding the CAR. For example, T cells within populations of PBMCs may be specifically activated by stimulation in vitro with peptides representing specific antigens, in the presence of IL-7 and IL-15.

Introducing nucleic acid(s)/vector(s) into a cell may comprise transduction, e.g. retroviral transduction. Accordingly, in some embodiments the nucleic acid(s) is/are comprised in a viral vector(s), or the vector(s) is/are a viral vector(s). Transduction of immune cells with viral vectors is described e.g. in Simmons and Alberola-lla, Methods Mol Biol. (2016) 1323:99-108, which is hereby incorporated by reference in its entirety.

Agents may be employed to enhance the efficiency of transduction. Hexadimethrine bromide (polybrene) is a cationic polymer which is commonly used to improve transduction, through neutralising charge repulsion between virions and sialic acid residues expressed on the cell surface. Other agents commonly used to enhance transduction include e.g. the poloxamer-based agents such as LentiBOOST (Sirion Biotech), Retronectin (Takara), Vectofusin (Miltenyi Biotech) and also SureENTRY (Qiagen) and ViraDuctin (Cell Biolabs).

In some embodiments the methods comprise centrifuging the cells into which it is desired to introduce nucleic acid encoding the CAR in the presence of cell culture medium comprising viral vector comprising the nucleic acid (referred to in the art as ‘spinfection’).

In some embodiments, the methods comprises introducing a nucleic acid or vector according to the present disclosure by electroporation, e.g. as described in Koh et al., Molecular Therapy - Nucleic Acids (2013) 2, e114, which is hereby incorporated by reference in its entirety.

The methods generally comprise introducing a nucleic acid encoding a CAR into a cell, and culturing the cell under conditions suitable for expression of the nucleic acid/CAR by the cell. In some embodiments, the methods culturing T cells into which nucleic acid encoding a CAR has been introduced in order to expand their number. In some embodiments, the methods comprise culturing T cells into which nucleic acid encoding a CAR has been introduced in the presence of IL-7 and/or IL-15 (e.g. recombinant IL-7 and/or IL-15).

In some embodiments the methods further comprise purifying/isolating CAR-expressing T cells, e.g. from other cells (e.g. cells which do not express the CAR). Methods for purifying/isolating immune cells from heterogeneous populations of cells are well known in the art, and may employ e.g. FACS- or MACS- based methods for sorting populations of cells based on the expression of markers of the immune cells.

In some embodiments the methods purifying/isolating cells of a particular type, e.g. CAR-expressing CD8+ T cells, CAR-expressing CTLs).

In preferred embodiments, CD30-specific CAR-expressing T cells may be generated from T cells within populations of PBMCs by a process comprising: stimulating PBMCs with peptides, transducing the cells with a viral vector (e.g. a gamma-retroviral vector) encoding the CD30-specific CAR, and subsequently culturing the cells in the presence of IL-7 and IL-15.

Alternatively, in some embodiments, the PBMCs are activated using agonistic CD3 and CD28 antibodies.

A CD30-specific CAR-expressing T cell according to the present disclosure may display certain functional properties of a T cell in response to CD30, or in response a cell comprising/expressing CD30. In some embodiments, the properties are functional properties associated with effector T cells, e.g. cytotoxic T cells.

In some embodiments, a CD30-specific CAR-expressing T cell may display one or more of the following properties: cytotoxicity to a cell comprising/expressing CD30; proliferation, IFNy expression, CD107a expression, IL-2 expression, TNFa expression, perforin expression, granzyme expression, granulysin expression, and/or FAS ligand (FASL) expression in response to stimulation with CD30, or in response to exposure to a cell comprising/expressing CD30; anti-cancer activity (e.g. cytotoxicity to cancer cells, tumor growth inhibition, reduction of metastasis, etc.) against cancer comprising cells expressing CD30.

Cell proliferation/population expansion can be investigated by analysing cell division or the number of cells over a period of time. Cell division can be analysed, for example, by in vitro analysis of incorporation of 3H-thymidine or by CFSE dilution assay, e.g. as described in Fulcher and Wong, Immunol Cell Biol (1999) 77(6): 559-564, hereby incorporated by reference in entirety. Proliferating cells can also be identified by analysis of incorporation of 5-ethynyl-2'-deoxyuridine (EdU) by an appropriate assay, as described e.g. in Buck et al., Biotechniques. 2008 Jun; 44(7):927-9, and Sali and Mitchison, PNAS USA 2008 Feb 19; 105(7): 2415-2420, both hereby incorporated by reference in their entirety.

As used herein, “expression” may be gene or protein expression. Gene expression encompasses transcription of DNA to RNA, and can be measured by various means known to those skilled in the art, for example by measuring levels of mRNA by quantitative real-time PCR (qRT-PCR), or by reporter-based methods. Similarly, protein expression can be measured by various methods well known in the art, e.g. by antibody-based methods, for example by western blot, immunohistochemistry, immunocytochemistry, flow cytometry, ELISA, ELISPOT, or reporter-based methods.

Cytotoxicity and cell killing can be investigated, for example, using any of the methods reviewed in Zaritskaya et al., Expert Rev Vaccines (201 1 ), 9(6) :601 -616, hereby incorporated by reference in its entirety. Examples of in vitro assays of cytotoxicity/cell killing assays include release assays such as the 51 Cr release assay, the lactate dehydrogenase (LDH) release assay, the 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyl tetrazolium bromide (MTT) release assay, and the calcein-acetoxymethyl (calcein-AM) release assay. These assays measure cell killing based on the detection of factors released from lysed cells. Cell killing by a given cell type can be analysed e.g. by co-culturing the test cells with the given cell type, and measuring the number/proportion of cells viable/dead test cells after a suitable period of time.

Cells may be evaluated for anti-cancer activity by analysis in an appropriate in vitro assays or in vivo models of the relevant cancer.

Aspects and embodiments of the present disclosure relate particularly to EBV-specific immune cells. Accordingly, in some embodiments, the virus may be EBV, and the viral antigen(s) may be EBV antigen(s). Methods for generating/expanding populations of EBV-specific immune cells are described e.g. in WO 2013/0881 14 A1 , Lapteva and Vera, Stem Cells Int. (201 1 ): 434392, Straathof et al., Blood (2005) 105(5): 1898-1904, WO 2017/202478 A1 , WO 2018/052947 A1 and WO 2020/214479 A1 , all of which are hereby incorporated by reference in their entirety.

The methods involve steps in which T cells comprising T cell receptors (TCRs) specific for EBV antigen peptide:MHC complex are stimulated by APCs presenting the EBV antigen peptide:MHC complex for which the TCR is specific. The APCs are infected with virus encoding, or comprise/express the EBV antigen/peptide(s), and present the EBV antigen peptide in the context of an MHC molecule. Stimulation causes T cell activation, and promotes cell division (proliferation), resulting in generation and/or expansion of a population of T cells specific for the EBV antigen.

The methods typically comprise stimulating immune cells specific for a virus/viral antigen by contacting populations of immune cells with peptide(s) corresponding to EBV antigen(s) or APCs presenting peptide(s) corresponding to viral antigen(s). Such method steps may be referred to herein as “stimulations” or “stimulation steps”. Such method steps typically involve maintenance of the cells in culture in vitro/ex vivo, and may be referred to as “stimulation cultures”.

In some embodiments, the methods comprise one or more additional stimulation steps. That is, in some embodiments the methods comprise one or more further steps of re-stimulating the cells obtained by a stimulation step. Such further stimulation steps may be referred to herein as “re-stimulations” or “restimulation steps”. Such method steps typically involve expansion of the cells in culture in vitro/ex vivo, and may be referred to as “re-stimulation cultures”. It will be appreciated that “contacting” PBMCs (for stimulations) or populations of cells obtained by a stimulation step described herein (for re-stimulations) with peptide(s) corresponding to viral antigen(s) generally involves culturing the PBMCs/population of cells in vitro/ex vivo in cell culture medium comprising the peptide(s). Similarly, it will be appreciated that “contacting” PBMCs/populations of cells with APCs presenting peptide(s) corresponding to viral antigen(s) generally involves co-culturing the APCs and the PBMCs/population of cells in vitro/ex vivo in cell culture medium.

In some embodiments, the methods comprise contacting PBMCs with peptide(s) corresponding to viral antigen(s) (e.g. EBV antigen(s)). In such embodiments, APCs within the population of PBMCs (e.g. monocytes, dendritic cells, macrophages and B cells) internalise (e.g. by phagocytosis, pinocytosis), process and present the antigens on MHC class I molecules (cross-presentation) and/or MHC class II molecules, for subsequent activation of CD8+ and/or CD4+ T cells within the population of PBMCs.

A peptide which “corresponds to” a reference antigen comprises or consists of an amino acid sequence of the reference antigen. For example, a peptide “corresponding to” EBNA1 of EBV comprises or consists of a sequence of amino acids which is found within the amino acid sequence of EBNA1 (i.e. is a subsequence of the amino acid sequence of EBNA1 ). Peptides employed herein typically have a length of 5-30 amino acids, e.g. one of 5-25 amino acids, 10-20 amino acids, or 12-18 amino acids. In some embodiments, peptides have a length of one of 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids. In some embodiments, peptides have a length of about 15 amino acids. “Peptides” as used herein may refer to populations comprising non-identical peptides.

In some embodiments, the methods employ peptides corresponding to more than one antigen. In such embodiments, there is at least one peptide which corresponds to each of the antigens. For example, where the methods employ peptides corresponding to EBNA1 and LMP1 , the peptides comprise at least one peptide corresponding to EBNA1 , and at least one peptide corresponding to LMP1 . In a preferred embodiment, the peptides have a length of 15 amino acids, overlapping by 11 amino acids, and spanning the entire protein sequence of the EBV antigens of interest. In some embodiments, the EBV antigens include EBNA1 , LMP1 , LMP2, BARF1 , BZLF1 , BRLF1 , BMLF1 , BMRF1 , BNLF2a, BNLF2b, BMRF2 and BALF2.

In some embodiments the methods employ peptides corresponding to all or part of a reference antigen. Peptides corresponding to all of a given antigen cover the full length of the amino acid sequence of the antigen. That is to say that together, the peptides contain all of the amino acids of the amino acid sequence of the given antigen. Peptides corresponding to part of a given antigen cover part of the amino acid sequence of the antigen. In some embodiments where peptides cover part of the amino acid sequence of the antigen, the peptides together may cover e.g. greater than 10%, e.g. greater than one of 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the amino acid sequence of the antigen. In some embodiments the methods employ overlapping peptides. “Overlapping” peptides have amino acids, and more typically sequences of amino acids, in common. By way of illustration, a first peptide consists of an amino acid sequence corresponding to positions 1 to 15 of the amino acid sequence of EBNA1 , and a second peptide consists of an amino acid sequence corresponding to positions 5 to 20 of the amino acid sequence of EBNA1 . The first and second peptides are overlapping peptides corresponding to EBNA1 , overlapping by 11 amino acids. In some embodiments overlapping peptides overlap by one of 1 -20, 5-20, 8-15 or 10-12 amino acids. In some embodiments overlapping peptides overlap by one of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15 amino acids. In some embodiments overlapping peptides overlap by 11 amino acids.

In some embodiments, the methods employ peptides having a length of 8-30 amino acids, overlapping by 1-20 amino acids, corresponding to all or part of a given reference antigen.

In some embodiments, the methods employ peptides having a length of 15 amino acids, overlapping by 11 amino acids, corresponding to all of a given reference antigen. Mixtures of such peptides may be referred to herein as “pepmix peptide pools” or “pepmixes” for a given antigen. For example, “EBNA1 pepmix” used in Example 1 herein refers to a pool of 158, 15mer peptides overlapping by 11 amino acids, spanning the full length of the amino acid sequence for EBNA1 as shown in UniProt: P03211 -1 , v1 .

In some embodiments in accordance with various aspects of the present disclosure, “peptides corresponding to” a given viral antigen may be a pepmix for the antigen.

In particular embodiments, the methods employ peptides corresponding to one or more EBV antigens. In particular embodiments, the methods employ pepmixes for one or more EBV antigens. In some embodiments, the one or more EBV antigens are selected from: an EBV latent antigen, e.g. a type III latency antigen (e.g. EBNA1 , EBNA-LP, LMP1 , LMP2A, LMP2B, BARF1 , EBNA2, EBNA3A, EBNA3B or EBNA3C), a type II latency antigen (e.g. EBNA1 , EBNA-LP, LMP1 , LMP2A, LMP2B or BARF1 ), or a type I latency antigen, (e.g. EBNA1 or BARF1 ), an EBV lytic antigen, e.g. an immediate-early lytic antigen (e.g. BZLF1 , BRLF1 or BMRF1 ), an early lytic antigen (e.g. BMLF1 , BMRF1 , BXLF1 , BALF1 , BALF2, BARF1 , BGLF5, BHRF1 , BNLF2A, BNLF2B, BHLF1 , BLLF2, BKRF4, BMRF2, FU or EBNA1 -FUK), and a late lytic antigen (e.g. BALF4, BILF1 , BILF2, BNFR1 , BVRF2, BALF3, BALF5, BDLF3 or gp350).

In some embodiments in accordance with various aspects of the present disclosure, the one or more EBV antigens are or comprise EBV lytic antigens selected from BZLF1 , BRLF1 , BMLF1 , BMRF1 , BXLF1 , BALF1 , BALF2, BGLF5, BHRF1 , BNLF2A, BNLF2B, BHLF1 , BLLF2, BKRF4, BMRF2, BALF4, BILF1 , BILF2, BNFR1 , BVRF2, BALF3, BALF5 and BDLF3. In some embodiments the one or more EBV antigens are or comprise EBV lytic antigens selected from BZLF1 , BRLF1 , BMLF1 , BMRF1 , BALF2, BNLF2A, BNLF2B, BMRF2 and BDLF3.

In some embodiments the one or more EBV antigens are or comprise EBV latent antigens selected from EBNA1 , EBNA-LP, EBNA2, EBNA3A, EBNA3B, EBNA3C, BARF1 , LMP1 , LMP2A and LMP2B. In some embodiments the one or more EBV antigens are or comprise EBV latent antigens selected from EBNA1 , LMP1 , LMP2A and LMP2B.

In some embodiments, the one or more EBV antigens are selected from: EBNA1 , LMP1 , LMP2, BARF1 , BZLF1 , BRLF1 , BMLF1 , BMRF1 , BMRF2, BALF2, BNLF2A and BNLF2B.

In some embodiments, the methods employ peptides corresponding to EBNA1 , LMP1 , LMP2, BARF1 , BZLF1 , BRLF1 , BMLF1 , BMRF1 , BMRF2, BALF2, BNLF2A and BNLF2B. In some embodiments, the methods employ pepmixes for EBNA1 , LMP1 , LMP2, BARF1 , BZLF1 , BRLF1 , BMLF1 , BMRF1 , BMRF2, BALF2, BNLF2A and BNLF2B.

In some embodiments, the methods comprise contacting PBMCs (e.g. PBMCs depleted of CD45RA- positive cells) with peptide(s) corresponding to EBNA1 , LMP1 , LMP2, BARF1 , BZLF1 , BRLF1 , BMLF1 , BMRF1 , BMRF2, BALF2, BNLF2A and BNLF2B. In some embodiments, the methods comprise contacting PBMCs (e.g. PBMCs depleted of CD45RA-positive cells) with pepmixes for EBNA1 , LMP1 , LMP2, BARF1 , BZLF1 , BRLF1 , BMLF1 , BMRF1 , BMRF2, BALF2, BNLF2A and BNLF2B.

In some embodiments, the methods comprise contacting the population of cells obtained by a stimulation step described herein with peptide(s) corresponding to viral antigen(s). In such embodiments, APCs within the population of cells (e.g. dendritic cells, macrophages and B cells) internalise (e.g. by phagocytosis, pinocytosis), process and present the antigens on MHC class I molecules (crosspresentation) and/or MHC class II molecules, for subsequent re-stimulation of CD8+ and/or CD4+ T cells within the population of cells.

In some embodiments, the methods comprise contacting PBMCs with APCs presenting peptide(s) corresponding to viral antigen(s). In some embodiments, the methods comprise contacting the population of cells obtained by a stimulation step described herein with APCs presenting peptide(s) corresponding to viral antigen(s).

In some embodiments, the methods comprise contacting PBMCs with EBV-LCLs. Production of EBV- specific immune cells by stimulating PBMCs with EBV-LCLs is described e.g. in Straathof et al., Blood (2005) 105(5): 1898-1904, which is incorporated by reference hereinabove.

EBV-LCLs may be prepared by infection of PBMCs with EBV, and collecting the immortalized EBV infected cells after long-term culture, e.g. as described in Hui-Yuen et al., J Vis Exp (2011 ) 57: 3321 , and Hussain and Mulherkar, Int J Mol Cell Med (2012) 1 (2): 75-87 (both hereby incorporated by reference in their entirety). EBV-specific T cells may be prepared by co-culture of PBMCs isolated from blood samples from healthy donors with autologous, irradiated EBV-LCLs.

Co-culture of T cells and APCs in stimulations and re-stimulations is performed in cell culture medium.

The cell culture medium can be any cell culture medium in which T cells and APCs according to the present disclosure can be maintained in culture in vitro/ex vivo. Culture medium suitable for use in the culture of lymphocytes is well known to the skilled person, and includes, for example, RPMI-1640 medium, AIM-V medium, Iscoves medium, etc.

In some embodiments, cell culture medium may comprise RPMI-1640 medium (e.g. Advanced RPMI- 1640 medium) and/or Click’s medium (also known as Eagle’s Ham’s amino acids (EHAA) medium). The compositions of these media are well known to the skilled person. The formulation of RPMI-1640 medium is described in e.g. Moore et al., JAMA (1967) 199:519-524, and the formulation of Click’s medium is described in Click et al., Cell Immunol (1972) 3:264-276. RPMI-1640 medium can be obtained from e.g. ThermoFisher Scientific, and Click’s medium can be obtained from e.g. Sigma-Aldrich (Catalog No. C5572). Advanced RPMI-1640 medium can be obtained from e.g. ThermoFisher Scientific (Catalog No. 12633012).

In some embodiments, the methods involve culturing PBMCs that have been contacted with peptide(s) corresponding to viral antigen(s) (e.g. EBV antigen(s)), or in the presence of APCs presenting peptide(s) corresponding to viral antigen(s), in cell culture medium comprising RPMI-1640 medium and Click’s medium. In some embodiments, the methods involve culturing the population of cells obtained by a stimulation step described herein that have been contacted with peptide(s) corresponding to viral antigen(s), or in the presence of APCs presenting peptide(s) corresponding to viral antigen(s), in cell culture medium comprising RPMI-1640 medium and Click’s medium.

In some embodiments the cell culture medium comprises (by volume) 25-65% RPMI-1640 medium, and 25-65% Click’s medium. In some embodiments the cell culture medium comprises 30-60% RPMI-1640 medium, and 30-60% Click’s medium. In some embodiments the cell culture medium comprises 35-55% RPMI-1640 medium, and 35-55% Click’s medium. In some embodiments the cell culture medium comprises 40-50% RPMI-1640 medium, and 40-50% Click’s medium. In some embodiments the cell culture medium comprises 45% RPMI-1640 medium, and 45% Click’s medium. In particular embodiments, the cell culture medium comprises 47.5% RPMI-1640 medium, and 47.5% Click’s medium.

In some embodiments, the cell culture medium may comprise one or more cell culture medium additives. Cell culture medium additives are well known to the skilled person, and include , growth factor-rich additives such as serum (e.g. human serum, fetal bovine serum (FBS), human platelet lysate, bovine serum albumin (BSA)), L-glutamine, cytokines/growth factors, etc.

In some embodiments, the cell culture medium comprises (by volume) 2.5-20% (e.g. 5%) growth factorrich additive, e.g. 5-20% FBS, e.g. 7.5-15% FBS, or 10% FBS. In some embodiments, the cell culture medium comprises 0.5-5% GlutaMax, e.g. 1 % GlutaMax. In some embodiments, the cell culture medium comprises 0.5-5% Pen/Strep, e.g. 1 % Pen/Strep.

In particular embodiments, the cell culture medium comprises human platelet lysate. In some embodiments, the cell culture medium comprises (by volume) 1 -20% (e.g. 5%) human platelet lysate, e.g. 2.5-20% human platelet lysate, e.g. 2.5-15%, 2.5-10%, or 5% human platelet lysate. Human platelet lysate can be obtained from e.g. Sexton Biotechnologies.

In particular embodiments, the cell culture medium comprises L-glutamine. In particular embodiments, the cell culture medium comprises 0.5-10 mM L-glutamine, e.g. 1 -5 mM L-glutamine, e.g. 2 mM L-glutamine.

APCs according to the present disclosure may be professional APCs. Professional APCs are specialised for presenting antigens to T cells; they are efficient at processing and presenting MHC-peptide complexes at the cell surface, and express high levels of costimulatory molecules. Professional APCs include dendritic cells (DCs), macrophages, and B cells. Non-professional APCs are other cells capable of presenting MHC-peptide complexes to T cells, in particular MHC Class l-peptide complexes to CD8+ T cells.

In some embodiments the APC is an APC capable of cross-presentation on MHC class I antigen following its internalisation by the APC (e.g. taken-up by endocytosis/phagocytosis). Cross-presentation on MHC class I of internalized antigens to CD8+ T cells is described e.g. in Alloatti et al., Immunological Reviews (2016), 272(1 ): 97-108, which is hereby incorporated by reference in its entirety. APCs capable of crosspresentation include e.g. dendritic cells (DCs), macrophages, B cells and sinusoidal endothelial cells.

As explained herein, in some embodiments APCs for stimulating immune cells specific for viral antigen(s) are comprised within the population of cells (e.g. PBMCs) comprising the immune cells specific for viral antigen(s), from which populations of cells specific for viral antigen(s) are to be expanded. In such embodiments, APCs may be e.g. monocytes, dendritic cells, macrophages, B cells or any other cell type within the population of cells which is capable of presenting antigen(s) to immune cells specific for viral antigen(s).

In some embodiments the methods employ APCs that have been modified to express/comprise viral antigen(s)/peptide(s) thereof. In some embodiments, the APCs may present peptide(s) corresponding to viral antigen(s) as a result of having been contacted with the peptide(s), and having internalised them. In some embodiments, APCs may have been “pulsed” with the peptide(s), which generally involves culturing APCs in vitro in the presence of the peptide(s), for a period of time sufficient for the APCs to internalise the peptide(s).

In some embodiments the APCs may present peptide(s) corresponding to viral antigen(s) as a result of expression of nucleic acid encoding the antigen within the cell. APCs may comprise nucleic acid encoding viral antigen(s) as a consequence of their having been infected with the virus (e.g. in the case of EBV- infected B cells, e.g. LCLs). APCs may comprise nucleic acid encoding viral antigen(s) as a consequence of nucleic acid encoding the antigen(s) having been introduced into the cell, e.g. via transfection, transduction, electroporation, etc. Nucleic acid encoding viral antigen(s) may be provided in a plasmid/vector. In some embodiments, APCs are selected from activated T cells (ATCs), dendritic cells, B cells (including e.g. LCLs), and artificial antigen presenting cells (aAPCs) such as those described in Neal et al., J Immunol Res Ther (2017) 2(1 ):68-79 and Turtle and Riddell Cancer J. (2010) 16(4):374-381 .

In some embodiments APCs are autologous with respect to the population of cells with which they are to be co-cultured for the generation/expansion of populations of immune cells comprising immune cells specific for viral antigen(s). That is, in some embodiments the APCs are from (or are derived from cells obtained from) the same subject as the subject from which the population of cells with which they are to be co-cultured were obtained.

The use of polyclonal activated T cells (ATCs) as APCs and methods for preparing ATCs are described e.g. in Ngo et al., J Immunother. (2014) 37(4) :193-203, incorporated by reference hereinabove. Briefly, ATCs can be generated by non-specifically activating T cells in vitro by stimulating PBMCs with agonist anti-CD3 and agonist anti-CD28 antibodies, in the presence of IL-2, or IL-7 and IL-15.

Dendritic cells may be generated according to methods well known in the art, e.g. as described in Ngo et al., J Immunother. (2014) 37(4) :193-203. Dendritic cells may be prepared from monocytes which may be obtained by CD14 selection from PBMCs. The monocytes may be cultured in cell culture medium causing their differentiation to immature dendritic cells, which may comprise e.g. IL-4 and GM-CSF. Immature dendritic cells may be matured by culture in the presence of IL-6, IL -1 p, TNFa, PGE2, GM-CSF and IL-4.

LCLs may be generated according to methods well known in the art, e.g. as described in Hui-Yuen et al., J Vis Exp (201 1 ) 57: 3321 , and Hussain and Mulherkar, Int J Mol Cell Med (2012) 1 (2): 75-87, both hereby incorporated by reference in their entirety. Briefly, LCLs can be produced by incubation of PBMCs with concentrated cell culture supernatant of cells producing EBV, for example B95-8 cells, in the presence of cyclosporin A.

Artificial costimulatory cells (aCs) include e.g. K562cs cells, which are HLA-negative and cannot present antigen, but are engineered to express costimulatory molecules CD80, CD86, CD83 and 4-1 BBL (described e.g. in Suhoski et al., Mol Ther. (2007) 15(5):981 -8).

In some embodiments, a stimulation step comprises contacting PBMCs with peptide(s) corresponding to viral antigen(s). In some embodiments, a re-stimulation step comprises contacting immune cells specific for viral antigen(s) with autologous APCs presenting peptide(s) corresponding to viral antigen(s) together with a costimulatory cell line to provide costimulation. In some embodiments, a re-stimulation step comprises contacting immune cells specific for viral antigen(s) with ATCs presenting peptide(s) corresponding to viral antigen(s).

In some embodiments, the methods further employ agents for enhancing costimulation in stimulations and/or re-stimulations. Such agents include e.g. cells expressing costimulatory molecules (e.g. CD80, CD86, CD83 and/or 4-1 BBL), such as e.g. LCLs or K562cs cells. In some embodiments the cells expressing costimulatory molecules are HLA-negative, EBV replication-incompetent LCLs, which are also referred to as “universal LCLs” or “ULCLs”. ULCLs are described e.g. in US 2018/0250379 A1 .

Other examples of agents for enhancing costimulation include e.g. agonist antibodies specific for costimulatory receptors expressed by T cells (e.g. 4-1 BB, CD28, 0X40, ICOS, etc.), and costimulatory molecules capable of activating costimulatory receptors expressed by T cells (e.g. CD80, CD86, CD83, 4- 1 BBL, OX40L, ICOSL, etc.). Such agents may be provided e.g. immobilised on beads.

In some embodiments stimulations and/or re-stimulations according to the present disclosure employ ULCLs. ULCLs also express CD30, along with other costimulatory molecules. Although ULCLs express EBV antigens, they are not presented to T-cells since ULCLs do not express MHC class I or class II molceules. Thus ULCLs are useful for, stimulation of CD30.CAR EBVSTs through the CAR via CD30, and also costimulation for the in vitro/ex vivo expansion of CD30.CAR EBVSTs, without stimulating alloreactive T-cells.

In some embodiments, the ULCLs are employed as cells providing antigenic stimulation (e.g. CD30 stimulation). In some embodiments, the ULCLs are employed as cells providing costimulation. In some embodiments, the ULCLs are employed as cells providing antigenic stimulation and costimulation. In some embodiments, the ULCLs are irradiated (e.g. at 100 gray).

In particular embodiments, the methods of the present disclosure comprise culturing immune cells specific for viral antigen(s) in the presence of ULCLs. In particular embodiments, the methods of the present disclosure comprise a restimulation step comprising culturing immune cells specific for viral antigen(s) in the presence of ULCLs. In some embodiments, ULCLs (e.g. irradiated ULCLs) may be employed in co-cultures with immune cells specific for viral antigen(s) at a ratio of immune cells specific for viral antigen(s) to ULCLs between 1 :1 and 1 :10, e.g. one of 1 :1 .5, 1 :2, 1 :2.5, 1 :3, 1 :3.5, 1 :4, 1 :4.5, 1 :5, 1 :5.5, 1 :6, 1 :6.5, 1 :7, 1 :7.5 or 1 :8. In some embodiments, ULCLs (e.g. irradiated ULCLs) may be employed in co-cultures with immune cells specific for viral antigen(s) at a ratio of immune cells specific for viral antigen(s) to ULCLs between 1 :2 and 1 :5, e.g. one of 1 :2, 1 :2.5, 1 :3, 1 :3.5, 1 :4, 1 :4.5 or 1 :5. In some embodiments, the ratio of immune cells specific for viral antigen(s) to ULCLs is ~1 :3.

In particular embodiments, the methods of the present disclosure comprise culturing virus-specific immune cells comprising/expressing a CAR described herein (or comprising/expressing nucleic acid encoding such a CAR) in the presence of ULCLs. In particular embodiments, the methods of the present disclosure comprise a restimulation step comprising culturing virus-specific immune cells comprising/expressing a CAR described herein (or comprising/expressing nucleic acid encoding such a CAR) in the presence of ULCLs. In some embodiments, ULCLs (e.g. irradiated ULCLs) may be employed in co-cultures with virus-specific immune cells comprising/expressing a CAR described herein (or comprising/expressing nucleic acid encoding such a CAR) at a ratio of virus-specific immune cells comprising/expressing a CAR described herein (or comprising/expressing nucleic acid encoding such a CAR) to ULCLs between 1 :1 and 1 :10, e.g. one of 1 :1 .5, 1 :2, 1 :2.5, 1 :3, 1 :3.5, 1 :4, 1 :4.5, 1 :5, 1 :5.5, 1 :6, 1 :6.5, 1 :7, 1 :7.5 or 1 :8. In some embodiments, ULCLs (e.g. irradiated ULCLs) may be employed in cocultures with virus-specific immune cells comprising/expressing a CAR described herein (or comprising/expressing nucleic acid encoding such a CAR) at a ratio of virus-specific immune cells comprising/expressing a CAR described herein (or comprising/expressing nucleic acid encoding such a CAR) to ULCLs between 1 :2 and 1 :5, e.g. one of 1 :2, 1 :2.5, 1 :3, 1 :3.5, 1 :4, 1 :4.5 or 1 :5. In some embodiments, the ratio of virus-specific immune cells comprising/expressing a CAR described herein (or comprising/expressing nucleic acid encoding such a CAR) to ULCLs is ~1 :3.

In some embodiments, a re-stimulation step comprises contacting immune cells specific for viral antigen(s) with ATCs presenting peptide(s) corresponding to viral antigen(s) in the presence of ULCLs.

Contacting of populations of immune cells with peptide(s) corresponding to viral antigen(s), or APCs presenting peptide(s) corresponding to viral antigen(s), may be performed in the presence of one or more cytokines, to facilitate T cell activation and proliferation. In some embodiments stimulations are performed in the presence of one or more of IL-7, IL-15, IL-6, IL-12, IL-4, IL-2 and/or IL-21 . It will be appreciated that the cytokines are added exogenously to the culture, and additional to cytokines that are produced by the cells in culture. In some embodiments the added cytokines are recombinantly-produced cytokines.

Accordingly, in some embodiments the methods involve culturing PBMCs that have been contacted with peptide(s) corresponding to viral antigen(s), or in the presence of APCs presenting peptide(s) corresponding to viral antigen(s), in the presence of one or more of IL-7, IL-15, IL-6, IL-12, IL-4, IL-2 and/or IL-21 .

In some embodiments culture is in the presence of IL-7, IL-15, IL-6, IL-12, IL-4, IL-2 and/or IL-21 . In some embodiments culture is in the presence of IL-7, IL-15, IL-6 and/or IL-12. In some embodiments culture is in the presence of IL-7 and/or IL-15.

In some embodiments the final concentration of IL-7 in the culture is 1 -100 ng/ml, e.g. one of 2-50 ng/ml, 5-20 ng/ml or 7.5-15 ng/ml. In some embodiments the final concentration of IL-7 in the culture is about 10 ng/ml.

In some embodiments the final concentration of IL-15 in the culture is 1 -100 ng/ml, e.g. one of 2-50 ng/ml, 5-20 ng/ml or 7.5-15 ng/ml. In some embodiments the final concentration of IL-15 in the culture is about 10 ng/ml. In some embodiments the final concentration of IL-15 in the culture is 10-1000 ng/ml, e.g. one of 20-500 ng/ml, 50-200 ng/ml or 75-150 ng/ml. In some embodiments the final concentration of IL-15 in the culture is about 100 ng/ml.

In some embodiments the final concentration of IL-6 in the culture is 10-1000 ng/ml, e.g. one of 20-500 ng/ml, 50-200 ng/ml or 75-150 ng/ml. In some embodiments the final concentration of IL-6 in the culture is about 100 ng/ml. In some embodiments the final concentration of IL-12 in the culture is 1 -100 ng/ml, e.g. one of 2-50 ng/ml, 5-20 ng/ml or 7.5-15 ng/ml. In some embodiments the final concentration of IL-12 in the culture is 10 ng/ml.

In some embodiments the final concentration of IL-7 is 1 -100 ng/ml (e.g. one of 2-50 ng/ml, 5-20 ng/ml or 7.5-15 ng/ml, e.g. 10 ng/ml), and the final concentration of IL-15 is 1 -100 ng/ml (e.g. one of 2-50 ng/ml, 5- 20 ng/ml or 7.5-15 ng/ml, e.g. about 10 ng/ml).

In some embodiments the final concentration of IL-7 is 1 -100 ng/ml (e.g. one of 2-50 ng/ml, 5-20 ng/ml or 7.5-15 ng/ml, e.g. 10 ng/ml), and the final concentration of IL-15 is 10-1000 ng/ml (e.g. one of 20-500 ng/ml, 50-200 ng/ml or 75-150 ng/ml, e.g. about 100 ng/ml).

In some embodiments the final concentration of IL-7 is 1 -100 ng/ml (e.g. one of 2-50 ng/ml, 5-20 ng/ml or 7.5-15 ng/ml, e.g. 10 ng/ml), the final concentration of IL-6 is 10-1000 ng/ml (e.g. one of 20-500 ng/ml, 50-200 ng/ml or 75-150 ng/ml, e.g. about 100 ng/ml), the final concentration of IL-12 is 1 -100 ng/ml (e.g. one of 2-50 ng/ml, 5-20 ng/ml or 7.5-15 ng/ml, e.g. 10 ng/ml), and the final concentration of IL-15 is 1 -100 ng/ml (e.g. one of 2-50 ng/ml, 5-20 ng/ml or 7.5-15 ng/ml, e.g. 10 ng/ml).

In some embodiments the final concentration of IL-7 in a stimulation culture is 1 -100 ng/ml (e.g. one of 2- 50 ng/ml, 5-20 ng/ml or 7.5-15 ng/ml, e.g. 10 ng/ml), and the final concentration of IL-15 in a stimulation culture is 10-1000 ng/ml (e.g. one of 20-500 ng/ml, 50-200 ng/ml or 75-150 ng/ml, e.g. about 100 ng/ml).

In some embodiments the final concentration of IL-7 in a stimulation culture is 1 -100 ng/ml (e.g. one of 2- 50 ng/ml, 5-20 ng/ml or 7.5-15 ng/ml, e.g. 10 ng/ml), the final concentration of IL-6 in a stimulation culture is 10-1000 ng/ml (e.g. one of 20-500 ng/ml, 50-200 ng/ml or 75-150 ng/ml, e.g. about 100 ng/ml), the final concentration of IL-12 in a stimulation culture is 1-100 ng/ml (e.g. one of 2-50 ng/ml, 5-20 ng/ml or 7.5-15 ng/ml, e.g. 10 ng/ml), and the final concentration of IL-15 in a stimulation culture is 1 -100 ng/ml (e.g. one of 2-50 ng/ml, 5-20 ng/ml or 7.5-15 ng/ml, e.g. 10 ng/ml).

In some embodiments the final concentration of IL-7 in a re-stimulation culture is 1 -100 ng/ml (e.g. one of 2-50 ng/ml, 5-20 ng/ml or 7.5-15 ng/ml, e.g. 10 ng/ml), and the final concentration of IL-15 in a restimulation culture is 10-1000 ng/ml (e.g. one of 20-500 ng/ml, 50-200 ng/ml or 75-150 ng/ml, e.g. about 100 ng/ml).

Stimulations and re-stimulations according to the present disclosure typically involve co-culture of T cells and APCs for a period of time sufficient for APCs to stimulate the T cells, and for the T cells to undergo cell division.

In some embodiments, the methods involve culturing PBMCs that have been contacted with peptide(s) corresponding to viral antigen(s), or in the presence of APCs presenting peptide(s) corresponding to viral antigen(s), for a period of one of at least 1 hour, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 4 days, 5 days, 6 days, or at least 7 days. In some embodiments, culture is for a period of 24 hours to 20 days, e.g. one of 48 hours to 14 days, 3 days to 12 days, 4 to 11 days, or 6 to 10 days or 7 to 9 days.

In some embodiments, the methods involve culturing the population of cells obtained by a stimulation step described herein that have been contacted with peptide(s) corresponding to viral antigen(s), or in the presence of APCs presenting peptide(s) corresponding to viral antigen(s), for a period of one of at least 1 hour, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 4 days, 5 days, 6 days, or at least 7 days. In some embodiments, culture is for a period of 24 hours to 20 days, e.g. one of 48 hours to 14 days, 3 days to 12 days, 4 to 11 days, or 6 to 10 days or 7 to 9 days.

Stimulations and re-stimulations may be ended by separating the cells in culture from the media in which they have been cultured, or diluting the culture, e.g. by the addition of cell culture medium. In some embodiments, the methods comprise a step of collecting the cells at the end of the stimulation or restimulation culture. In some embodiments, a re-stimulation step may be established by adding cell culture medium (and any other additives as described herein) in an amount appropriate to achieve the desired percentages/concentrations of cell culture medium, conditioned media (and any additives) for the restimulation step.

At the end of the culture period of a given stimulation or re-stimulation step, the cells may be collected and separated from the cell culture supernatant. The cells may be collected by centrifugation, and the cell culture supernatant may be separated from the cell pellet. The cell pellet may then be re-suspended in cell culture medium, e.g. for a re-stimulation step. In some embodiments, the cells may undergo a washing step after collection. A washing step may comprise re-suspending the cell pellet in isotonic buffer such as phosphate-buffered saline (PBS), collecting the cells by centrifugation, and discarding the supernatant.

Methods for generating and/or expanding populations of immune cells specific for viral antigen(s) typically involve more than a single stimulation step. There is no upper limit to the number of stimulation steps which may be performed. In some embodiments the methods comprise more than 2, 3, 4 or 5 stimulation steps. In some embodiments, the methods comprise one of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, or 15 stimulation steps. The stimulation steps in a method may be different to one another.

In some embodiments, the PBMCs employed in the methods are depleted of CD45RA-positive cells. That is, in some embodiments, the PBMCs are “CD45RA-positive cell-depleted PBMCs”, or are “CD45RA- negative PBMCs”. Depletion of CD45RA-positive cells is intended to reduce the number of NK cells and/or regulatory T cells and/or naive T-cells in the populations of cells generated/expanded.

In some embodiments, the methods comprise a step of depleting PBMCs of CD45RA-positive cells, e.g. prior to a stimulation step. In some embodiments, the methods comprise a step of depleting the cells obtained by a stimulation step according to the present disclosure of CD45RA-positive cells, e.g. prior to a re-stimulation step. Depletion of CD45RA-positive cells can be achieved by any suitable method, such as by magnetic-activated cell sorting (MACS), for example using Miltenyi® Biotec columns and magnetic anti-CD45RA antibody-coated beads.

In some embodiments, the population of cells used to derive APCs employed in the methods is depleted of CD45RA-positive cells. That is, in some embodiments, the population of cells used to derive APCs is a “CD45RA-positive cell-depleted” or “CD45RA-negative” population. For example, in embodiments wherein ATCs are employed as APCs, the ATCs may be derived from a population of CD45RA-positive cell-depleted PBMCs, or from a population of CD45RA-negative PBMCs.

The nucleic acid may be introduced into the cells by methods well known in the art, such as transduction, transfection, electroporation, etc. In some embodiments the nucleic acid is introduced into the cells via transduction using a viral vector (e.g. a retroviral vector) comprising the nucleic acid.

Aspects and embodiments of the methods described herein comprise modifying an immune cell described herein (e.g. a virus-specific immune cell described herein) to express/comprise a CAR according to the present disclosure.

Aspects and embodiments of the methods described herein comprise modifying an immune cell described herein (e.g. a virus-specific immune cell described herein) to express/comprise nucleic acid encoding a CAR according to the present disclosure.

Such methods typically comprise introducing nucleic acid encoding a CAR into an immune cell.

Immune cells (e.g. virus-specific immune cells) may be modified to comprise/express a CAR or nucleic acid encoding a CAR described herein according to methods that are well known to the skilled person. The methods generally comprise nucleic acid transfer for permanent (stable) or transient expression of the transferred nucleic acid.

Any suitable genetic engineering platform may be used to modify a cell according to the present disclosure. Suitable methods for modifying a cell include the use of genetic engineering platforms such as gammaretroviral vectors, lentiviral vectors, adenovirus vectors, DNA transfection, transposon-based gene delivery and RNA transfection, for example as described in Maus et al., Annu Rev Immunol (2014) 32:189-225, hereby incorporated by reference in its entirety. In some embodiments, modifying a cell to comprise a CAR or nucleic acid encoding a CAR comprises transducing a cell with a viral vector comprising nucleic acid encoding the CAR. In some embodiments, the methods of the present disclosure employ a retrovirus encoding a CAR described herein.

Methods also include those described e.g. in Wang and Riviere Mol Ther Oncolytics. (2016) 3:16015, which is hereby incorporated by reference in its entirety.

The methods generally comprise introducing a nucleic acid/plurality of nucleic acids encoding a vector/plurality of vectors comprising such nucleic acid(s), into a cell. In some embodiments, the methods additionally comprise culturing the cell under conditions suitable for expression of the nucleic acid(s) or vector(s) by the cell. In some embodiments, the methods are performed in vitro. Suitable methods for introducing nucleic acid(s)/vector(s) into cells include transduction, transfection and electroporation.

In some embodiments, introducing nucleic acid(s)/vector(s) into a cell comprises transduction, e.g. retroviral transduction. Accordingly, in some embodiments the nucleic acid(s) is/are comprised in a viral vector(s), or the vector(s) is/are a viral vector(s). Transduction of immune cells with viral vectors is described e.g. in Simmons and Alberola-lla, Methods Mol Biol. (2016) 1323:99-108, which is hereby incorporated by reference in its entirety.

Agents may be employed in the methods of the present disclosure to enhance the efficiency of transduction. Hexadimethrine bromide (polybrene) is a cationic polymer which is commonly used to improve transduction, through neutralising charge repulsion between virions and sialic acid residues expressed on the cell surface. Other agents commonly used to enhance transduction include e.g. the poloxamer-based agents such as LentiBOOST (Sirion Biotech), Retronectin (Takara), Vectofusin (Miltenyi Biotech) and also SureENTRY (Qiagen) and ViraDuctin (Cell Biolabs).

In particular embodiments, the methods of the present disclosure employ Vectofusin-1 (Miltenyi Biotec Cat No. 170-076-165) in the transduction of cells with a vector/nucleic acid encoding a CAR described herein. In some embodiments, the methods comprise contacting retrovirus encoding a CAR described herein with Vectofusin-1 , and contacting cells to be transduced with the retrovirus with the mixture comprising retrovirus and Vectofusin-1 .

In some embodiments, the methods comprise centrifuging the cells into which it is desired to introduce nucleic acid encoding the CAR in the presence of cell culture medium comprising viral vector comprising the nucleic acid (referred to in the art as ‘spinfection’).

In some embodiments, the methods comprise introducing a nucleic acid or vector according to the present disclosure by electroporation, e.g. as described in Koh et al., Molecular Therapy - Nucleic Acids (2013) 2, e1 14, which is hereby incorporated by reference in its entirety.

In some embodiments, the methods further comprise purifying/isolating CAR-expressing and/or virusspecific immune cells, e.g. from other cells (e.g. cells which are not specific for the virus, and/or cells which do not express the CAR). Methods for purifying/isolating immune cells from heterogeneous populations of cells are well known in the art, and may employ e.g. FACS- or MACS-based methods for sorting populations of cells based on the expression of markers of the immune cells. In some embodiments the method is for purifying/isolating cells of a particular type, e.g. virus-specific T cells (e.g. virus-specific CD8+ T cells, virus-specific CTLs), or CAR-expressing virus-specific T cells (e.g. CAR- expressing virus-specific CD8+ T cells, CAR-expressing virus-specific CTLs).

The present disclosure also provides cells obtained or obtainable by the methods described herein, and populations thereof.

Virus-specific T cells

The present disclosure concerns CD30-specific CAR-expressing T cells. It will be appreciated that where cells are referred to herein in the singular (i.e. “a/the cell”), pluralities/populations of such cells are also contemplated.

In some embodiments, the CD30-specific CAR-expressing T cells are virus-specific T cells. “Virusspecific T cells” as used herein refers to a T cell which is specific for a virus. A virus-specific T cell expresses/comprises a receptor (preferably a T cell receptor) capable of recognising a peptide of an antigen of a virus (e.g. when presented by an MHC molecule). The virus-specific T cell may express/comprise such a receptor as a result of expression of endogenous nucleic acid encoding such antigen receptor, or as a result of having been engineered to express such a receptor. The virus-specific T cell preferably expresses/comprises a TCR specific for a peptide of an antigen of a virus.

In some embodiments, the T cell is a CD3+, CD4+ T cell. In some embodiments, the T cell is a CD3+, CD8+ T cell. In some embodiments, the T cell is a T helper cell (TH cell). In some embodiments, the T cell is a cytotoxic T cell (e.g. a cytotoxic T lymphocyte (CTL)).

A virus-specific T cell may display certain functional properties of a T cell in response to the viral antigen for which the T cell is specific, or in response a cell comprising/expressing the virus/antigen. In some embodiments, the properties are functional properties associated with effector T cells, e.g. cytotoxic T cells.

In some embodiments, a virus-specific T cell may display one or more of the following properties: cytotoxicity to a cell comprising/expressing the virus /the viral antigen for which the T cell is specific; proliferation, IFNy expression, CD107a expression, IL-2 expression, TNFa expression, perforin expression, granzyme expression, granulysin expression, and/or FAS ligand (FASL) expression in response to stimulation with the virus/the viral antigen for which the T cell is specific, or in response to exposure to a cell comprising/expressing the virus /the viral antigen for which the T cell is specific.

Virus-specific T cells express/comprise a TCR capable of recognising a peptide of the viral antigen for which the T cell is specific when presented by the appropriate MHC molecule. Virus-specific T cells may be CD4+ T cells and/or CD8+ T cells. In some embodiments, the virus-specific T cell is specific for Epstein-Barr virus. In some embodiments, the CD30-specific CAR-expressing T cells are Epstein-Barr Virus-specific T cells (CD30.CAR-EBVSTs).

In some embodiments, the virus-specific immune cell may be specific for a peptide/polypeptide of an Epstein-Barr virus.

A T cell which is specific for an antigen of a virus may be referred to herein as a virus-specific T cell (VST). A T cell which is specific for an antigen of a particular virus may be described as being specific for the relevant virus; for example, a T cell which is specific for an antigen of EBV may be referred to as an EBV-specific T cell, or “EBVST”.

Accordingly, in some embodiments the virus-specific T cell is an Epstein-Barr virus-specific T cell (EBVST).

In some preferred embodiments, the virus-specific immune cell is specific for a peptide/polypeptide of an EBV antigen. In preferred embodiments the virus-specific immune cell is an Epstein-Barr virus-specific T cell (EBVST).

EBV virology is described e.g. in Stanfield and Luftiq, F1000Res. (2017) 6:386 and Odumade et al., Clin Microbiol Rev (2011 ) 24(1 ):193-209, both of which are hereby incorporated by reference in their entirety.

EBV infects epithelial cells via binding of viral protein BMFR2 to p1 integrins, and binding of viral protein gH/gL with integrins avp6 and avp8. EBV infects B cells through interaction of viral glycoprotein gp350 with CD21 and/or CD35, followed by interaction of viral gp42 with MHC class II. These interactions trigger fusion of the viral envelope with the cell membrane, allowing the virus to enter the cell. Once inside, the viral capsid dissolves and the viral genome is transported to the nucleus.

EBV has two modes of replication; latent and lytic. The latent cycle does not result in production of new infectious virions, and can take place in place B cells and epithelial cells. The EBV genomic circular DNA resides in the latently infected cell nucleus as an episome and is copied by the host cell’s DNA polymerase. In latency, only a fraction of EBV's genes are expressed, in one of three different patterns known as latency programs, which produce distinct sets of viral proteins and RNAs. The latent cycle is described e.g. in Amon and Farrell, Reviews in Medical Virology (2004) 15(3): 149-56, which is hereby incorporated by reference in its entirety.

EBNA1 protein and non-coding RNA EBER are expressed in each of latency programs l-lll. Latency programs II and III further involve expression of EBNALP, LMP1 , LMP2A and LMP2B proteins, and latency program III further involves expression of EBNA2, EBNA3A, EBNA3B and EBNA3C. EBNA1 is multifunctional, and has roles in gene regulation, extrachromosomal replication, and maintenance of the EBV episomal genome through positive and negative regulation of viral promoters (Duellman et al., J Gen Virol. (2009); 90(Pt 9): 2251-2259). EBNA2 is involved in the regulation of latent viral transcription and contributes to the immortalization of cells infected with EBV (Kempkes and Ling, Curr Top Microbiol Immunol. (2015) 391 :35-59). EBNA-LP is required for transformation of native B cells, and recruits transcription factors for viral replication (Szymula et al., PLoS Pathog.

(2018);14(2):e1006890). EBNA3A, 3B and 3C interact with RBPJ to influence gene expression, contributing to survival and growth of infected cells (Wang et al., J Virol. (2016) 90(6):2906-2919). LMP1 regulates expression of genes involved in B cell activation (Chang et al., J. Biomed. Sci. (2003) 10(5): 490-504). LMP2A and LMP2B inhibit normal B cell signal transduction by mimicking the activated B cell receptor (Portis and Longnecker, Oncogene (2004) 23(53): 8619-8628). EBERs form ribonucleoprotein complexes with host cell proteins, and are proposed to have roles in cell transformation.

The latent cycle can progress according to any of latency programs I to III in B cells, and usually progresses from III to II to I. Upon infection of a resting naive B cell, EBV enters latency program III. Expression of latency III genes activates the B cell, which becomes a proliferating blast. EBV then typically progresses to latency II by restricting expression to a subset of genes, which cause differentiation of the blast to a memory B cell. Further restriction of gene expression causes EBV to enter latency I. EBNA1 expression allows EBV to replicate when the memory B cell divides. In epithelial cells, only latency II occurs.

In primary infection, EBV replicates in oropharyngeal epithelial cells and establishes Latency III, II, and I infections in B-lymphocytes. EBV latent infection of B-lymphocytes is necessary for virus persistence, subsequent replication in epithelial cells, and release of infectious virus into saliva. EBV Latency III and II infections of B-lymphocytes, Latency II infection of oral epithelial cells, and Latency II infection of NK- or T cell can result in malignancies, marked by uniform EBV genome presence and gene expression.

Latent EBV in B cells can be reactivated to switch to lytic replication. The lytic cycle results in the production of infectious virions and can take place in place B cells and epithelial cells, and is reviewed e.g. by Kenney in Chapter 25 of Arvin et al., Human Herpesviruses: Biology, Therapy and Immunoprophylaxis; Cambridge University Press (2007), which is hereby incorporated by reference in its entirety.

Lytic replication requires the EBV genome to be linear. The latent EBV genome is episomal, and so it must be linearised for lytic reactivation. In B cells, lytic replication normally only takes place after reactivation from latency.

Immediate-early lytic gene products such as BZFL1 and BRLF1 act as transactivators, enhancing their own expression, and the expression of later lytic cycle genes. Early lytic gene products have roles in viral replication (e.g. EBV DNA polymerase catalytic component BALF5; DNA polymerase processivity factor BMRF1 , DNA binding protein BALF2, helicase BBLF4, primase BSLF1 , and primase-associated protein BBLF2/3) and deoxynucleotide metabolism (e.g. thymidine kinase BXLF1 , dUTPase BORF2). Other early lytic gene products act transcription factors (e.g. BMRF1 , BRRF1 ), have roles in RNA stability and processing (e.g. BMLF1 ), or are involved in immune evasion (e.g. BHRF1 , which inhibits apoptosis).

Late lytic gene products are traditionally classed as those expressed after the onset of viral replication. They generally encode structural components of the virion such as nucleocapsid proteins, as well as glycoproteins which mediate EBV binding and fusion (e.g. gp350/220, gp85, gp42, gp25). Other late lytic gene products have roles in immune evasion; BCLF1 encodes a viral homologue of IL-10, and BALF1 encodes a protein with homology to the anti-apoptotic protein Bcl2.

An “EBV-specific T cell” as used herein refers to a T cell which is specific for Epstein-Barr virus (EBV). An EBV-specific T cell expresses/comprises a receptor (preferably a T cell receptor) capable of recognising a peptide of an antigen of EBV (e.g. when presented by an MHC molecule). The EBV-specific T cell preferably expresses/comprises a TCR specific for a peptide of an EBV antigen presented by MHC class I.

In some embodiments, the EBV-specific T cell is a CD3+, CD4+ T cell. In some embodiments, the T cell is a CD3+, CD8+ T cell. In some embodiments, the T cell is a T helper cell (TH cell)). In some embodiments, the T cell is a cytotoxic T cell (e.g. a cytotoxic T lymphocyte (CTL)).

An EBV-specific T cell may display certain functional properties of a T cell in response to the EBV antigen for which the T cell is specific, or in response a cell comprising/expressing EBV (e.g. a cell infected with EBV) or the relevant EBV antigen. In some embodiments, the properties are functional properties associated with effector T cells, e.g. cytotoxic T lymphocytes (CTLs).

In some embodiments, an EBV-specific T cell may display one or more of the following properties: cytotoxicity to a cell comprising/expressing EBV/the EBV antigen for which the T cell is specific; proliferation, IFNy expression, CD107a expression, IL-2 expression, TNFa expression, perforin expression, granzyme expression, granulysin expression, and/or FAS ligand (FASL) expression in response to stimulation with EBV/the EBV antigen for which the T cell is specific, or in response to exposure to a cell comprising/expressing EBV/the EBV antigen for which the T cell is specific.

EBV-specific T cells preferably express/comprise a TCR capable of recognising a peptide of the EBV antigen for which the T cell is specific when presented by the appropriate MHC molecule. EBV-specific T cells may be CD4+ T cells and/or CD8+ T cells. A T cell specific for EBV may be specific for any EBV antigen, e.g. an EBV antigen described herein. A population of immune cell specific for EBV, or a composition comprising a plurality of immune cells specific for EBV, may comprise immune cells specific for one or more EBV antigens.

In some embodiments, an EBV antigen is an EBV latent antigen, e.g. a type III latency antigen (e.g. EBNA1 , EBNA-LP, LMP1 , LMP2A, LMP2B, BARF1 , EBNA2, EBNA3A, EBNA3B or EBNA3C), a type II latency antigen (e.g. EBNA1 , EBNA-LP, LMP1 , LMP2A, LMP2B or BARF1 ), or a type I latency antigen, (e.g. EBNA1 or BARF1 ). In some embodiments, an EBV antigen is an EBV lytic antigen, e.g. an immediate-early lytic antigen (e.g. BZLF1 , BRLF1 or BMRF1 ), an early lytic antigen (e.g. BMLF1 , BMRF1 , BXLF1 , BALF1 , BALF2, BARF1 , BGLF5, BHRF1 , BNLF2A, BNLF2B, BHLF1 , BLLF2, BKRF4, BMRF2, FU or EBNA1 -FUK), or a late lytic antigen (e.g. BALF4, BILF1 , BILF2, BNFR1 , BVRF2, BALF3, BALF5, BDLF3 or gp350).

Allogeneic CD30-specific CAR-expressing T cells

Aspects of the present disclosure employ CD30-specific chimeric antigen receptor (CAR)-expressing T cells that are allogeneic to the subject. In some embodiments, the CD30-specific CAR-expressing T cells are Epstein-Barr Virus-specific T cells (CD30.CAR-EBVSTs).

The present disclosure provides a method of eliminating alloreactive T cells in a subject with a CD30- positive cancer, comprising administering a dose of allogeneic CD30-specific chimeric antigen receptor (CAR)-expressing T cells to the subject, wherein a first part of the dose is administered at a first time point and the remaining part of the dose is administered at a second time point, wherein the first and second time points are 2 to 4 days apart.

The present disclosure also provides a composition comprising allogeneic CD30-specific chimeric antigen receptor (CAR)-expressing T cells for use in a method of eliminating alloreactive T cells in a subject with a CD30-positive cancer, wherein the method comprises administering a dose of CD30-specific chimeric antigen receptor (CAR)-expressing T cells to the subject, wherein the dose is administered in two parts at two time points, wherein a first part of the dose is administered at a first time point and the remaining part of the dose is administered at a second time point, wherein the first and second time points are 2 to 4 days apart. In addition, the present disclosure provides the use of a composition comprising allogeneic CD30-specific chimeric antigen receptor (CAR)-expressing T cells in the manufacture of a medicament for use in a method of eliminating alloreactive T cells in a subject with a CD30-positive cancer, wherein the method comprises administering a dose of CD30-specific chimeric antigen receptor (CAR)-expressing T cells to the subject, wherein the dose is administered in two parts at two time points, wherein a first part of the dose is administered at a first time point and the remaining part of the dose is administered at a second time point, wherein the first and second time points are 2 to 4 days apart.

Alloreactive T cells comprise TCRs capable of recognising non-self MHC molecules (i.e. allogeneic MHC), and initiating an immune response thereto. Alloreactive T cells may display one or more of the following properties in response to a cell expressing a non-self MHC molecule: cell proliferation, growth factor (e.g. IL-2) expression, cytotoxic/effector factor (e.g. IFNy, granzyme, perforin, granulysin, CD107a, TNFa, FASL) expression and/or cytotoxic activity.

“Alloreactivity” and an “alloreactive immune response” as used herein refers to an immune response directed against a cell/tissue/organ which is genetically non-identical to the effector immune cell. An effector immune cell may display alloreactivity or an alloreactive immune response to cells - or tissues/organs comprising cells - expressing non-self MHC/HLA molecules (/.e. MHC/HLA molecules which are non-identical to the MHC/HLA molecules encoded by the effector immune cells).

“MHC mismatched” and “HLA mismatched” subjects as referred to herein are subjects having MHC/HLA genes encoding non-identical MHC/HLA molecules. In some embodiments the MHC mismatched or HLA mismatched subjects have MHC/HLA genes encoding non-identical MHC class I a and/or MHC class II molecules. “MHC matched” and “HLA matched” subjects as referred to herein are subjects having MHC/HLA genes encoding identical MHC/HLA molecules. In some embodiments the MHC matched or HLA matched subjects have MHC/HLA genes encoding identical MHC class I a and/or MHC class II molecules.

Where a cell/tissue/organ is referred to herein as being allogeneic with respect to a reference subject/treatment, the cell/tissue/organ is from obtained/derived from cells/tissue/organ of a subject other than the reference subject. In some embodiments, allogeneic material comprises MHC/HLA genes encoding MHC/HLA molecules (e.g. MHC class I a and/or MHC class II molecules) which are non- identical to the MHC/HLA molecules (e.g. MHC class I a and/or MHC class II molecules) encoded by the MHC/HLA genes of the reference subject.

Where a cell/tissue/organ is referred to herein as being allogeneic with respect to a treatment, the cell/tissue/organ is from obtained/derived from cells/tissue/organ of a subject other than the subject to be treated. In some embodiments, allogeneic material comprises MHC/HLA genes encoding MHC/HLA molecules (e.g. MHC class I a and/or MHC class II molecules) which are non-identical to the MHC/HLA molecules (e.g. MHC class I a and/or MHC class II molecules) encoded by the MHC/HLA genes of the subject to be treated.

Where a cell/tissue/organ is referred to herein as being allogeneic with respect to a reference subject, cell/tissue/organ is genetically non-identical to the reference subject, or derived/obtained from a genetically non-identical subject. Where a cell/tissue/organ is referred to herein as being allogeneic in the context of a treatment of a subject, the cell/tissue/organ is genetically non-identical to the subject to be treated, or derived/obtained from a genetically non-identical subject. Allogeneic cell/tissue/organs may comprise MHC/HLA genes encoding MHC/HLA molecules (e.g. MHC class I a and/or MHC class II molecules) which are non-identical to the MHC/HLA molecules (e.g. MHC class I a and/or MHC class II molecules) encoded by the MHC/HLA genes of the reference subject. In some embodiments, immune cells specific for a virus expressing/comprising a CAR described herein (or expressing/comprising nucleic acid encoding such a CAR) to be administered to a subject in accordance with the methods of the present disclosure are selected based on the HLA/MHC profile of the subject to be treated.

In some embodiments, the cells to be administered to the subject are selected based on their being HLA/MHC matched with respect to the subject. In some embodiments, the cells to be administered to the subject are selected based on their being a near or complete HLA/MHC match with respect to the subject.

As used herein, HLA/MHC alleles may be determined to ‘match’ when they encode polypeptides having the same amino acid sequence. That is, the ‘match’ is determined at the protein level, irrespective of the possible presence of synonymous differences in the nucleotide sequences encoding the polypeptides and/or differences in the non-coding regions.

Cells which are ‘HLA matched’ with respect to a reference subject may be: (i) an 8/8 match across HLA-

A, -B, -C, and -DRB1 ; or (ii) a 10/10 match across HLA-A, -B, -C, -DRB1 and -DQB1 ; or (iii) a 12/12 match across HLA-A, -B, -C, -DRB1 , -DQB1 and -DPB1 . Cells which are ‘a near or complete HLA match’ with respect to a reference subject may be: (i) a >4/8 (i.e. 4/8, 5/8, 6/8, 7/8 or 8/8) match across HLA-A, -

B, -C, and -DRB1 ; or (ii) a >5/10 (i.e. 5/10, 6/10, 7/10, 8/10, 9/10 or 10/10) match across HLA-A, -B, -C, - DRB1 and -DQB1 ; or (iii) a >6/12 (i.e. 6/12, 7/12, 8/12, 9/12, 10/12, 11/12 or 12/12) match across HLA-A, -B, -C, -DRB1 , -DQB1 and -DPB1 . Cells may be partially HLA matched with respect to a reference subject, where the cells may be (i) <4/8 (i.e. 1/8, 2/8, 3/8 or 4/8) match across HLA-A, -B, -C, and DRB-1 , or (ii) a <5/10 (i.e. 1/10, 2/10, 3/10, 4/10 or 5/10) match across HLA-A, -B, -C, -DRB1 and -DQB1 ; or (iii) a <6/12 (i.e. 1/12, 2/12, 3/12, 4/12, 5/12, or 6/12) match across HLA-A, -B, -C, -DRB1 , -DQB1 and -DPB1.

Administration of cells to a subject which are a near or complete HLA match (irrespective of their being of allogeneic origin) can be advantageous, especially in the context of administration of immune cells specific for a virus expressing/comprising a CAR described herein (or expressing/comprising nucleic acid encoding such a CAR) for the treatment of a disease/condition caused by, or associated with, infection with the virus for which the immune cells are specific. In such instances, presentation of viral antigens by cells of the host to the administered cells (through their native TCRs) would be expected to increase their activation, proliferation and survival in vivo, and consequently improve their therapeutic efficacy.

One common problem facing “off-the-shelf” T-cell therapy products is graft rejection mediated by the patient’s own alloreactive T cells. The administration of allogeneic CD30-specific chimeric antigen receptor (CAR)-expressing cells (e.g. CD30.CAR-EBVSTs) as a split dose according to the methods disclosed herein is advantageous as it allows for the elimination of alloreactive T cells. Administration of a first dose of CD30-specific CAR-expressing cells causes alloreactive T cells (which are initially negative for expression of CD30) to activate and travel to the lymph nodes to proliferate, upregulate CD30 expression and acquire effector (e.g. killing) functions against the donor CD30-specific CAR-expressing cells. The CD30.CAR-EBVSTs will not be eliminated at this point because the alloreactive T cells lack effector function. Following administration of a second dose of CD30-specific CAR-expressing cells 2-4 days after the first dose, the CD30-specific CAR-expressing T cells will travel to both the CD30-positive tumor and to the site of alloreactive T cell proliferation, because the activated T cells will secrete chemokines that recruit effector cells. The donor CD30-specific CAR-expressing T cells will therefore be able to eliminate the alloreactive T cells that now express CD30.

Thus, the CD30.CAR-EBVSTs will (i) eliminate the alloreactive T cells they elicit in allogeneic hosts, and (ii) persist for sufficient time and with the requisite activity to eliminate CD30-positive cancer.

As disclosed herein, “elimination” or “depletion” of alloreactive T cells may be total or partial. Elimination of alloreactive T cells may be determined using, for example, flow cytometry of T cells derived from the subject, or mixed lymphocyte reactions (MLRs).

Elimination or depletion of alloreactive immune cells may result in, e.g. a 2-fold, 10-fold, 100-fold, 1000- fold, 10000-fold or greater reduction in the quantity of alloreactive immune cells in a subject.

Accordingly, the present disclosure provides a method of eliminating alloreactive T cells in a subject with a CD30-positive cancer, comprising administering a dose of allogeneic CD30-specific chimeric antigen receptor (CAR)-expressing T cells to the subject, wherein a first part of the dose is administered at a first time point and the remaining part of the dose is administered at a second time point, wherein the first and second time points are 2 to 4 days apart.

The present disclosure also provides a composition comprising allogeneic CD30-specific chimeric antigen receptor (CAR)-expressing T cells for use in a method of eliminating alloreactive T cells in a subject with a CD30-positive cancer, wherein the method comprises administering a dose of CD30-specific chimeric antigen receptor (CAR)-expressing T cells to the subject, wherein the dose is administered in two parts at two time points, wherein a first part of the dose is administered at a first time point and the remaining part of the dose is administered at a second time point, wherein the first and second time points are 2 to 4 days apart.

The present disclosure also provides the use of a composition comprising allogeneic CD30-specific chimeric antigen receptor (CAR)-expressing T cells in the manufacture of a medicament for use in a method of eliminating alloreactive T cells in a subject with a CD30-positive cancer, wherein the method comprises administering a dose of CD30-specific chimeric antigen receptor (CAR)-expressing T cells to the subject, wherein the dose is administered in two parts at two time points, wherein a first part of the dose is administered at a first time point and the remaining part of the dose is administered at a second time point, wherein the first and second time points are 2 to 4 days apart.

Aspects of the present disclosure employ lymphodepleting chemotherapy. As used herein, “lymphodepleting chemotherapy” refers to treatment with a chemotherapeutic agent which results in depletion of lymphocytes (e.g. T cells, B cells, NK cells, NKT cells or innate lymphoid cell (ILCs), or precursors thereof) within the subject to which the treatment is administered. A “lymphodepleting chemotherapeutic agent” refers to a chemotherapeutic agent which results in depletion of lymphocytes.

Lymphodepleting chemotherapy and its use in methods of treatment by adoptive cell transfer are described e.g. in Klebanoff et al., Trends Immunol. (2005) 26(2):111 -7 and Muranski et al., Nat Clin Pract Oncol. (2006) (12) :668-81 , both of which are hereby incorporated by reference in their entirety. The aim of lymphodepleting chemotherapy is to deplete the recipient subject’s endogenous lymphocyte population.

In the context of treatment of disease by adoptive transfer of immune cells, lymphodepleting chemotherapy is typically administered prior to adoptive cell transfer, to condition the recipient subject to receive the adoptively transferred cells. Lymphodepleting chemotherapy is thought to promote the persistence and activity of adoptively transferred cells by creating a permissive environment, e.g. through elimination of cells expressing immunosuppressive cytokines, and creating the ‘lymphoid space’ and homeostatic cytokines, e.g. IL-7 and IL-15 required for expansion and activity of adoptively transferred lymphoid cells.

Chemotherapeutic agents commonly used in lymphodepleting chemotherapy include e.g. fludarabine, bendamustine, cyclophosphamide and pentostatin.

Aspects and embodiments of the present disclosure are particularly concerned with lymphodepleting chemotherapy comprising administration of fludarabine, cyclophosphamide and/or bendamustine. In particular embodiments, lymphodepleting chemotherapy according to the present disclosure comprises administration of fludarabine and cyclophosphamide. In some embodiments, the lymphodepleting chemotherapy comprises cyclophosphamide and bendamustine. In some embodiments, the lymphodepleting chemotherapy comprises fludarabine and bendamustine.

Fludarabine is a purine analog that inhibits DNA synthesis by interfering with ribonucleotide reductase and DNA polymerase. It is often employed as a chemotherapeutic agent for the treatment of leukemia (particularly chronic lymphocytic leukemia, acute myeloid leukemia, acute lymphocytic leukemia) and lymphoma (particularly non-Hodgkin’s Lymphoma). Fludarabine may be administered intravenously or orally.

Cyclophosphamide and bendamustine are alkylating agents which cause intra-strand and inter-strand cross-links between DNA bases. They are often employed as a chemotherapeutic agent for the treatment of chronic lymphocytic leukemia, multiple myeloma and non-Hodgkin’s Lymphoma. Bendamustine and cyclophosphamide are typically administered intravenously. Methods of treatment

The present disclosure provides methods for the treatment of CD30-positive cancer, compositions for use in such methods, and the use of compositions for the manufacture of medicaments for use in such methods.

The methods generally comprise administering CD30-specific CAR-expressing T cells to the subject. Specifically, the present disclosure provides a method of treating a CD30-positive cancer in a subject, comprising administering a dose of CD30-specific chimeric antigen receptor (CAR)-expressing T cells to the subject, wherein the dose is administered in two parts at two time points, wherein a first part of the dose is administered at a first time point and the remaining part of the dose is administered at a second time point, wherein the first and second time points are 2 to 4 days apart.

The present disclosure also provides CD30-specific CAR-expressing T cells (e.g. a composition of such cells) for use in a method of treating a CD30-positive cancer, wherein the method comprises administering a dose of CD30-specific chimeric antigen receptor (CAR)-expressing T cells to a subject, wherein the dose is administered in two parts at two time points, wherein a first part of the dose is administered at a first time point and the remaining part of the dose is administered at a second time point, wherein the first and second time points are 2 to 4 days apart. The present disclosure also provides the use of CD30-specific CAR-expressing T cells (e.g. a composition of such cells) in the manufacture of a medicament for use in a method of treating a CD30-positive cancer, wherein the method comprises administering CD30-specific CAR-T cells to the subject, wherein the dose is administered in two parts at two time points, wherein a first part of the dose is administered at a first time point and the remaining part of the dose is administered at a second time point, wherein the first and second time points are 2 to 4 days apart.

The CD30-specific CAR-expressing T cells may be administered as a dose which is split between two separate time points. The method may comprise administering a dose of CD30-specific chimeric antigen receptor (CAR)-expressing T cells to the subject, wherein the dose is split between two separate time points and wherein the time points are 2 to 4 days apart. The present disclosure also provides CD30- specific CAR-expressing T cells (e.g. a composition of such cells) for use in a method of treating a CD30- positive cancer, wherein the method comprises administering CD30-specific CAR-T cells to the subject, wherein the dose is split between two separate time points and wherein the time points are 2 to 4 days apart. The present disclosure also provides the use of CD30-specific CAR-expressing T cells (e.g. a composition of such cells) in the manufacture of a medicament for use in a method of treating a CD30- positive cancer, wherein the method comprises administering CD30-specific CAR-T cells to the subject, wherein the dose is split between two separate time points and wherein the time points are 2 to 4 days apart.

In some embodiments, the methods comprise administering a lymphodepleting chemotherapy to a subject having a CD30-positive cancer, and subsequently administering CD30-specific CAR-expressing T cells to the subject. The present disclosure also provides a lymphodepleting chemotherapeutic agent (e.g. fludarabine, cyclophosphamide and/or bendamustine) for use in a method of treating a CD30-positive cancer, wherein the method comprises: (i) administering a lymphodepleting chemotherapy (e.g. comprising administering fludarabine, cyclophosphamide and/or bendamustine) to the subject, and (ii) subsequently administering CD30-specific CAR-T cells to the subject. The present disclosure also provides the use of a lymphodepleting chemotherapeutic agent (e.g. fludarabine, cyclophosphamide and/or bendamustine) in the manufacture of a medicament for use in a method of treating a CD30-positive cancer, wherein the method comprises: (i) administering a lymphodepleting chemotherapy (e.g. comprising administering fludarabine, cyclophosphamide and/or bendamustine) to the subject, and (ii) subsequently administering CD30-specific CAR-T cells to the subject.

The present disclosure also provides fludarabine for use in a method of treating a CD30-positive cancer, wherein the method comprises: (i) administering a lymphodepleting chemotherapy comprising administering fludarabine (e.g. a lymphodepleting chemotherapy comprising administering fludarabine and cyclophosphamide) to the subject, and (ii) subsequently administering CD30-specific CAR-T cells to the subject. The present disclosure also provides the use of fludarabine in the manufacture of a medicament for use in a method of treating a CD30-positive cancer, wherein the method comprises: (i) administering a lymphodepleting chemotherapy comprising administering fludarabine (e.g. a lymphodepleting chemotherapy comprising administering fludarabine and cyclophosphamide) to the subject, and (ii) subsequently administering CD30-specific CAR-T cells to the subject.

The present disclosure also provides cyclophosphamide for use in a method of treating a CD30-positive cancer, wherein the method comprises: (i) administering a lymphodepleting chemotherapy comprising administering cyclophosphamide (e.g. a lymphodepleting chemotherapy comprising administering fludarabine and cyclophosphamide) to the subject, and (ii) subsequently administering CD30-specific CAR-T cells to the subject. The present disclosure also provides the use of cyclophosphamide in the manufacture of a medicament for use in a method of treating a CD30-positive cancer, wherein the method comprises: (i) administering a lymphodepleting chemotherapy comprising administering cyclophosphamide (e.g. a lymphodepleting chemotherapy comprising administering fludarabine and cyclophosphamide) to the subject, and (ii) subsequently administering CD30-specific CAR-T cells to the subject.

The present disclosure also provides bendamustine for use in a method of treating a CD30-positive cancer, wherein the method comprises: (i) administering a lymphodepleting chemotherapy comprising administering bendamustine (e.g. a lymphodepleting chemotherapy comprising cyclophosphamide and bendamustine, or a lymphodepleting chemotherapy comprising fludarabine and bendamustine), and (ii) subsequently administering CD30-specific CAR-T cells to the subject. The present disclosure also provides the use of bendamustine in the manufacture of a medicament for use in a method of treating a CD30-positive cancer, wherein the method comprises: (i) administering a lymphodepleting chemotherapy comprising administering bendamustine (e.g. a lymphodepleting chemotherapy comprising administering cyclophosphamide and bendamustine or a lymphodepleting chemotherapy comprising administering fludarabine and bendamustine) to the subject, and (ii) subsequently administering CD30-specific CAR-T cells to the subject.

The present disclosure also provides the combination of fludarabine and cyclophosphamide (e.g. a pharmaceutical composition or combination comprising fludarabine and cyclophosphamide) for use in a method of treating a CD30-positive cancer, wherein the method comprises: (i) administering a lymphodepleting chemotherapy comprising administering fludarabine and cyclophosphamide to the subject, and (ii) subsequently administering CD30-specific CAR-T cells to the subject. The present disclosure also provides the use of the combination of fludarabine and cyclophosphamide (e.g. a pharmaceutical composition or combination comprising fludarabine and cyclophosphamide) in the manufacture of a medicament for use in a method of treating a CD30-positive cancer, wherein the method comprises: (i) administering a lymphodepleting chemotherapy comprising administering fludarabine and cyclophosphamide to the subject, and (ii) subsequently administering CD30-specific CAR-T cells to the subject.

The present disclosure also provides the combination of cyclophosphamide and bendamustine (e.g. a pharmaceutical composition or combination comprising cyclophosphamide and bendamustine) for use in a method of treating a CD30-positive cancer, wherein the method comprises: (i) administering a lymphodepleting chemotherapy comprising administering cyclophosphamide and bendamustine to the subject, and (ii) subsequently administering CD30-specific CAR-T cells to the subject. The present disclosure also provides the use of the combination of cyclophosphamide and bendamustine (e.g. a pharmaceutical composition or combination comprising cyclophosphamide and bendamustine) in the manufacture of a medicament for use in a method of treating a CD30-positive cancer, wherein the method comprises: (i) administering a lymphodepleting chemotherapy comprising administering cyclophosphamide and bendamustine to the subject, and (ii) subsequently administering CD30-specific CAR-T cells to the subject.

The present disclosure also provides the combination of fludarabine and bendamustine (e.g. a pharmaceutical composition or combination comprising fludarabine and bendamustine) for use in a method of treating a CD30-positive cancer, wherein the method comprises: (i) administering a lymphodepleting chemotherapy comprising administering fludarabine and bendamustine to the subject, and (ii) subsequently administering CD30-specific CAR-T cells to the subject. The present disclosure also provides the use of the combination of fludarabine and bendamustine (e.g. a pharmaceutical composition or combination comprising fludarabine and bendamustine) in the manufacture of a medicament for use in a method of treating a CD30-positive cancer, wherein the method comprises: (i) administering a lymphodepleting chemotherapy comprising administering fludarabine and bendamustine to the subject, and (ii) subsequently administering CD30-specific CAR-T cells to the subject.

The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of the cancer to be treated, and the nature of the agent. Prescription of treatment, e.g. decisions on dosage etc., is within the responsibility of general practitioners and other medical doctors, and typically takes account of the cancer to be treated, the condition of the individual subject, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington’s Pharmaceutical Sciences, 20th Edition, 2000, pub. Lippincott, Williams & Wilkins.

For administration in accordance with the present disclosure, cells and chemotherapeutic agents are preferably formulated as medicaments or pharmaceutical compositions comprising pharmaceutically acceptable ingredients well known to those skilled in the art, including, but not limited to, pharmaceutically acceptable carriers, adjuvants, excipients, diluents, fillers, buffers, preservatives, anti-oxidants, lubricants, stabilisers, solubilisers, surfactants (e.g., wetting agents), masking agents, colouring agents, flavouring agents, and sweetening agents.

The term "pharmaceutically acceptable" as used herein pertains to compounds, ingredients, materials, compositions, dosage forms, etc., which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of the subject in question (e.g., human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. Each carrier, adjuvant, excipient, etc. must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation. Suitable carriers, adjuvants, excipients, etc. can be found in standard pharmaceutical texts, for example, Remington's Pharmaceutical Sciences, 18th edition, Mack Publishing Company, Easton, Pa., 1990; and Handbook of Pharmaceutical Excipients, 2nd edition, 1994.

The formulations may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing into association the relevant active agent with a carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active compound with carriers (e.g., liquid carriers, finely divided solid carrier, etc.), and then shaping the product, if necessary.

The cells and chemotherapeutic agents of the present disclosure may be formulated for a mode of administration which is acceptable in accordance with the agent and the cancer to be treated. For example, cells and chemotherapeutic agents according to the present invention may be formulated for intravascular administration, e.g. intravenous injection or infusion to a subject. Suitable formulations may comprise the selected agent in a sterile or isotonic medium.

A course of lymphodepleting chemotherapy in accordance with the present disclosure may comprise multiple administrations of one or more chemotherapeutic agents.

A course of lymphodepleting chemotherapy may comprise administering fludarabine and cyclophosphamide at a dose described herein, and for a number of days described herein. By way of illustration, a course of lymphodepleting chemotherapy may comprise administering fludarabine at a dose of 30 mg/m² per day for 3 consecutive days, and administering cyclophosphamide at a dose of 500 mg/m² per day for 3 consecutive days.

A course of lymphodepleting chemotherapy may comprise administering cyclophosphamide and bendamustine at a dose described herein, and for a number of days described herein. By way of illustration, a course of lymphodepleting chemotherapy may comprise administering cyclophosphamide at a dose of 500 mg/m² per day for 3 consecutive days, and administering bendamustine at a dose of 70 mg/m² per day, for 3 consecutive days.

A course of lymphodepleting chemotherapy may comprise administering fludarabine and bendamustine at a dose described herein, and for a number of days described herein. By way of illustration, a course of lymphodepleting chemotherapy may comprise administering fludarabine at a dose of 30 mg/m² per day for 3 consecutive days, and administering bendamustine at a dose of 70 mg/m² per day, for 3 consecutive days.

The day of administration of the final dose of a chemotherapeutic agent in accordance with a course of lymphodepleting chemotherapy may be considered to be the day of completion of the course of lymphodepleting chemotherapy.

In some embodiments, fludarabine is administered at a dose of 5 to 100 mg/m² per day, e.g. one of 15 to 90 mg/m² per day, 15 to 80 mg/m² per day, 15 to 70 mg/m² per day, 15 to 60 mg/m² per day, 15 to 50 mg/m² per day, 10 to 40 mg/m² per day, 5 to 60 mg/m² per day, 10 to 60 mg/m² per day, 15 to 60 mg/m² per day, 20 to 60 mg/m² per day or 25 to 60 mg/m² per day. In some embodiments, fludarabine is administered at a dose of 20 to 40 mg/m² per day, e.g. 25 to 35 mg/m² per day, e.g. about 30 mg/m² per day.

In some embodiments fludarabine is administered at a dose according to the preceding paragraph for more than one day and fewer than 14 consecutive days. In some embodiments, fludarabine is administered at a dose according to the preceding paragraph for one of 2 to 14 e.g. 2 to 13, 2 to 12, 2 to 11 , 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5 or 2 to 4 consecutive days. In some embodiments, fludarabine is administered at a dose according to the preceding paragraph for 2 to 6 consecutive days, e.g. 2 to 4 consecutive days, e.g. 3 consecutive days.

In some embodiments fludarabine is administered at a dose of 15 to 60 mg/m² per day, for 2 to 6 consecutive days, e.g. at a dose of 30 mg/m² per day, for 3 consecutive days.

In some embodiments, cyclophosphamide is administered at a dose of 250 to 1000 mg/m² per day, e.g. one of 250 to 750 mg/m² per day, 250 to 700 mg/m² per day, 250 to 650 mg/m² per day, 250 to 600 mg/m² per day, 250 to 550 mg/m² per day, 250 to 500 mg/m² per day, 300 to 1000 mg/m² per day, 350 to 1000 mg/m² per day, 400 to 1000 mg/m² per day, 500 to 1000 mg/m² per day, 550 to 1000 mg/m² per day, 600 to 1000 mg/m² per day, 650 to 1000 mg/m² per day, 700 to 1000 mg/m² per day, 750 to 1000 mg/m² per day, 800 to 1000 mg/m² per day, 850 to 1000 mg/m² per day, or 900 to 1000 mg/m² per day.

In some embodiments, cyclophosphamide is administered at a dose according to the preceding paragraph for more than one day and fewer than 14 consecutive days. In some embodiments, cyclophosphamide is administered at a dose according to the preceding paragraph for one of 2 to 14 e.g. 2 to 13, 2 to 12, 2 to 1 1 , 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5 or 2 to 4 consecutive days. In some embodiments, cyclophosphamide is administered at a dose according to the preceding paragraph for 2 to 6 consecutive days, e.g. 2 to 4 consecutive days, e.g. 3 consecutive days.

In some embodiments, cyclophosphamide is administered at a dose of 250 to 1000 mg/m² per day, for 2 to 6 consecutive days, e.g. at a dose of 500 mg/m² per day, for 3 consecutive days.

In some embodiments, bendamustine is administered at a dose of 10 to 200 mg/m² per day, e.g. one of 35 to 180 mg/m² per day, 35 to 160 mg/m² per day, 35 to 140 mg/m² per day, 35 to 120 mg/m² per day, 35 to 100 mg/m² per day, 35 to 80 mg/m² per day, 10 to 100 mg/m² per day, 15 to 100 mg/m² per day, 20 to 100 mg/m² per day, 25 to 100 mg/m² per day, 30 to 100 mg/m² per day, 35 to 100 mg/m² per day, 40 to 100 mg/m² per day, 45 to 100 mg/m² per day, 50 to 100 mg/m² per day, 55 to 100 mg/m² per day, 60 to 100 mg/m² per day, or 65 to 100 mg/m² per day.

In some embodiments, bendamustine is administered at a dose according to the preceding paragraph for more than one day and fewer than 14 consecutive days. In some embodiments, bendamustine is administered at a dose according to the preceding paragraph for one of 2 to 14 e.g. 2 to 13, 2 to 12, 2 to 11 , 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5 or 2 to 4 consecutive days. In some embodiments, bendamustine is administered at a dose according to the preceding paragraph for 2 to 6 consecutive days, e.g. 2 to 4 consecutive days, e.g. 3 consecutive days.

In some embodiments, bendamustine is administered at a dose of 35 to 140 mg/m² per day, for 2 to 6 consecutive days, e.g. at a dose of 70 mg/m² per day, for 3 consecutive days.

In some embodiments, the methods comprise administering fludarabine at a dose of 15 to 60 mg/m² per day (e.g. 30 mg/m² per day) and administering cyclophosphamide at a dose of 250 to 1000 mg/m² per day (e.g. 500 mg/m² per day), for 2 to 6 consecutive days (e.g. 3 consecutive days).

In some embodiments, the methods comprise administering cyclophosphamide at a dose of 250 to 1000 mg/m² (e.g. 500 mg/m² per day) and administering bendamustine at a dose of 35 to 140 mg/m² per day (e.g. 70 mg/m² per day), for 2 to 6 consecutive days (e.g. 3 consecutive days).

In some embodiments, the methods comprise administering fludarabine at a dose of 15 to 60 mg/m² per day (e.g. 30 mg/m² per day) and administering bendamustine at a dose of 35 to 140 mg/m² per day (e.g. 70 mg/m² per day), for 2 to 6 consecutive days (e.g. 3 consecutive days). In some embodiments, fludarabine and cyclophosphamide may be administered simultaneously or sequentially. In some embodiments, cyclophosphamide and bendamustine may be administered simultaneously or sequentially. In some embodiments, fludarabine and bendamustine may be administered simultaneously or sequentially. Simultaneous administration refers to administration together, for example as a pharmaceutical composition containing both agents (i.e. in a combined preparation), or immediately after one another, and optionally via the same route of administration, e.g. to the same artery, vein or other blood vessel. Sequential administration refers to administration of one of the agents followed after a given time interval by separate administration of the other agent. It is not required that the agents are administered by the same route, although this is the case in some embodiments.

In some embodiments of courses of lymphodepleting chemotherapy in accordance with the present disclosure, fludarabine and cyclophosphamide are administered on the same day or days. By way of illustration, in the example of a course of lymphodepleting chemotherapy comprising administering fludarabine at a dose of 30 mg/m² per day for 3 consecutive days, and administering cyclophosphamide at a dose of 500 mg/m² per day for 3 consecutive days, the fludarabine and cyclophosphamide may be administered on the same 3 consecutive days. In such an example, the course of lymphodepleting chemotherapy may be said to be completed on the final day of the 3 consecutive days on which fludarabine and cyclophosphamide are administered to the subject.

Lymphodepleting chemotherapy may be administered by intravenous infusion over an appropriate period of time. In some embodiments, a lymphodepleting chemotherapeutic agent may be administered by intravenous infusion over a period of 15 to 60 min, e.g. 20 to 40 min, e.g. about 30 min.

Aspects of the present disclosure also comprise administering CD30-specific CAR-expressing T cells to a subject having a CD30-positive cancer. The methods therefore involve adoptive cell transfer. In some embodiments, the methods comprise adoptive transfer of allogeneic CD30-specific chimeric antigen receptor (CAR)-expressing T cells.

Adoptive cell transfer generally refers to a process by which cells (e.g. immune cells) are obtained from a subject, typically by drawing a blood sample from which the cells are isolated. The cells are then typically modified and/or expanded, and then administered either to the same subject (in the case of adoptive transfer of autologous/autogeneic cells) or to a different subject (in the case of adoptive transfer of allogeneic cells). Adoptive cell transfer is typically aimed at providing a population of cells with certain desired characteristics to a subject, or increasing the frequency of such cells with such characteristics in that subject. Adoptive transfer may be performed with the aim of introducing a cell or population of cells into a subject, and/or increasing the frequency of a cell or population of cells in a subject.

Adoptive transfer of CD30-specific CAR-expressing T cells is described, for example, in Hornbach et al. J Immunol (2001 ) 167:6123-6131 , Ramos et al. J. Clin. Invest. (2017) 127(9):3462-3471 and WO 2015/028444 A1 , all of which are incorporated by reference hereinabove. The skilled person is able to determine appropriate reagents and procedures for adoptive transfer of such cells in accordance with the methods of the present disclosure by reference to these documents.

The present disclosure provides methods comprising administering a T cell comprising/expressing a CD30-specific CAR, or a T cell comprising/expressing nucleic acid encoding a CD30-specific CAR, to a subject.

In some embodiments, the methods comprise modifying a T cell to comprise/express a CD30-specific CAR. In some embodiments, the methods comprise modifying a T cell to comprise/express nucleic acid encoding a CD30-specific CAR.

In some embodiments, the methods comprise:

(a) modifying a T cell to express or comprise a CD30-specific CAR, or to express or comprise nucleic acid encoding a CD30-specific CAR; and

(b) administering T cell modified to express or comprise a CD30-specific CAR, or modified to express or comprise nucleic acid encoding a CD30-specific CAR, to a subject.

In some embodiments, the methods comprise:

(a) isolating or obtaining a population of immune cells comprising T cells (e.g. PBMCs);

(b) modifying a T cell to express or comprise a CD30-specific CAR, or to express or comprise nucleic acid encoding a CD30-specific CAR; and

(c) administering a T cell modified to express or comprise a CD30-specific CAR, or modified to express or comprise nucleic acid encoding a CD30-specific CAR, to a subject.

In some embodiments, the methods comprise:

(a) isolating or obtaining a population of immune cells comprising T cells (e.g. PBMCs) from a subject;

In some embodiments, the subject from which the population of immune cells comprising T cells (e.g. PBMCs) is isolated is the same subject to which cells are administered (i.e., adoptive transfer may be of autologous/autogeneic cells). In some embodiments, the subject from which the population of immune cells comprising T cells (e.g. PBMCs) is isolated is a different subject to the subject to which cells are administered (i.e., adoptive transfer may be of allogeneic cells).

In some embodiments the methods may comprise one or more of: obtaining a blood sample from a subject; isolating a population of immune cells comprising T cells (e.g. PBMCs) from a blood sample which has been obtained from a subject; culturing the immune cells in vitro or ex v/vo cell culture; modifying a T cell to express or comprise a CD30-specific CAR, or to express or comprise nucleic acid encoding a CD30-specific CAR (e.g. by transduction with a viral vector encoding such CAR, or a viral vector comprising such nucleic acid); culturing T cells modified to express or comprise a CD30-specific CAR, or modified to express or comprise nucleic acid encoding a CD30-specific CAR in in vitro or ex v/vo cell culture; collecting/isolating T cells modified to express or comprise a CD30-specific CAR, or modified to express or comprise nucleic acid encoding a CD30-specific CAR; formulating T cells modified to express or comprise a CD30-specific CAR, or modified to express or comprise nucleic acid encoding a CD30-specific CAR to a pharmaceutical composition, e.g. by mixing the cells with a pharmaceutically acceptable adjuvant, diluent, or carrier; administering T cells modified to express or comprise a CD30-specific CAR, or modified to express or comprise nucleic acid encoding a CD30-specific CAR, or a pharmaceutical composition comprising such cells, to a subject.

In some embodiments, the methods may additionally comprise treating the cells or subject to induce/enhance expression of CAR and/or to induce/enhance proliferation or survival of cells comprising/expressing the CAR.

In some embodiments, a blood sample may be obtained by venesection or leukapheresis, which are both well known to the skilled person. The total blood volume of a blood sample obtained by venesection is preferably between 100 ml to 500 ml, e.g. 150 ml to 300 ml, e.g. about 200 ml. Blood sample collection is preferably performed a sufficient period of time prior to planned administration of CD30-specific CAR- expressing T cells to a subject for the production of a sufficient quantity of CD30-specific CAR-expressing T cells for a dose to be administered to a subject. In some embodiments, a blood sample is obtained at 6 to 8 weeks prior to planned administration of CD30-specific CAR-expressing T cells to a subject.

In the methods of the present disclosure, CD30-specific CAR-expressing T cells are administered to the subject after lymphodepleting chemotherapy has been administered to the subject.

In some embodiments, CD30-specific CAR-expressing T cells are administered to a subject within a specified period of time following completion of a course of lymphodepleting chemotherapy, e.g. a course of lymphodepleting chemotherapy described herein. That is, CD30-specific CAR-expressing T cells are administered to a subject within a specified period of time following the day of administration of the final dose of a chemotherapeutic agent in accordance with administration of a lymphodepleting chemotherapy in accordance with the present disclosure.

In some embodiments, CD30-specific CAR-expressing T cells are administered to a subject within 1 to 28 days, e.g. one of 1 to 21 days, 1 to 14 days, 1 to 7 days, 2 to 7 days, 2 to 5 days, or 3 to 5 days of completion of a course of lymphodepleting chemotherapy described herein. In some embodiments, CD30-specific CAR-expressing T cells are administered to a subject within 2 to 14 days of completion of a course of lymphodepleting chemotherapy described herein. In some embodiments, CD30-specific CAR- expressing T cells are administered to a subject within 3 to 5 days of completion of a course of lymphodepleting chemotherapy described herein.

Dose of CAR-T cells

Administration of cells and chemotherapeutic agents in accordance with the methods of the present disclosure is preferably in a “therapeutically effective” amount, this being sufficient to show therapeutic benefit to the subject.

Administration of CD30-specific CAR-expressing T cells may be administered by intravenous infusion. Administration may be in a volume containing 0.5 to 6 x 10⁷ cells/ml, e.g. 1 to 3 x 10⁷ cells/ml.

The methods of the present disclosure typically comprise administering a dose of CD30-specific chimeric antigen receptor (CAR)-expressing T cells to the subject, wherein the dose is administered in two parts at two time points, wherein a first part of the dose is administered at a first time point and the remaining part of the dose (or second part of the dose) is administered at a second time point, wherein the first and second time points are 2 to 4 days apart.

The methods of the present disclosure may comprise administering a dose of CD30-specific chimeric antigen receptor (CAR)-expressing T cells to the subject, wherein the dose is split between two separate time points and wherein the time points are 2 to 4 days apart.

References to a “dose” may refer to the total amount of cells to be administered per treatment. Thus, each “treatment” may involve one dose, and that dose can be split between a plurality of time points, or administered at a plurality of time points, or administered as a plurality of “parts” at a plurality of time points. The “parts” may be referred to as, for example, a first part and a second part, or as a first part and a remaining part. Accordingly, each “dose” may be divided into a plurality of sub-doses, part-doses, or administrations of the dose.

The total amount of cells to be administered per treatment may also be referred to as a “total dose”. Thus, each “treatment” may involve one total dose. The total dose may comprise a first dose and a second dose which may be administered at a plurality of time points.

Accordingly, the term “dose” or “total dose” may be used to refer to a therapeutically useful amount or number of cells, rather than the number of cells administered in a single administration on a single day.

References to a dose being “split” may refer to a dose or total dose that is administered to the subject in two parts. For example, 50% of the dose or total dose may be administered on day 0 and the remaining 50% of the dose or total dose may be administered on day 2, 3 or 4. Alternatively, 40-60% of the dose or total dose may be administered on day 0 and the remaining 40-60% of the dose or total dose may be administered on day 2, 3 or 4. In some embodiments 40-60% of the dose or total dose is administered on day 0 and the remaining 40-60% of the dose or total dose is administered on day 3.

References to time points being “2 days apart” may mean, for example, that one time point (or the first time point) is on day 0 and the next time point (or second time point) is on day 2. References to time points being “3 days apart” may mean, for example, that one time point (or the first time point) is on day 0 and the next time point (or the second time point) is on day 3. References to time points being “4 days apart” may mean, for example, that one time point (or the first time point) is on day 0 and the next time point (or the second time point) is on day 4.

References to first and second doses being administered “2 days apart” may mean that the first dose is administered on day 0 and the second dose is administered on day 2. References to first and second doses being administered “3 days apart” may mean that the first dose is administered on day 0 and the second dose is administered on day 3. References to first and second doses being administered “4 days apart” may mean that the first dose is administered on day 0 and the second dose is administered on day 4.

References to “2 days apart”, “3 days apart” and “4 days apart”, etc. may mean that the time point is (or the dose is administered at) any time on that day, i.e. within the 24-hour period of that day. For example, a first dose may be administered at any time on day 0 and a second dose may be administered at any time on day 2, 3 or 4.

The methods of the present disclosure may comprise administering a total dose of CD30-specific chimeric antigen receptor (CAR)-expressing T cells to the subject, wherein the total dose comprises a first dose and a second dose, wherein the first and second dose are administered 2 to 4 days apart. In some embodiments, the first and second dose are administered 3 days apart.

In some embodiments, a first part of the dose of CD30-specific CAR-expressing T cells is administered at a first time point and the remaining part of the dose is administered at second time point, wherein the first and second time points are 2 to 4 days apart. In some embodiments, the first part of the dose and the remaining part of the dose are administered 3 days apart.

In some embodiments, CD30-specific CAR-expressing T cells are administered at a dose or total dose of 4 x 10⁷ cells/m² to 4 x 10⁸ cells/m², e.g. one of 4 x 10⁷ cells/m², 1 x 10⁸ cells/m², or 4 x 10⁸ cells/m². In some embodiments, CD30-specific CAR-expressing T cells are administered at a dose or total dose of 4 x 10⁷ cells/m². In some embodiments, CD30-specific CAR-expressing T cells are administered at a dose or total dose of 4 x 10⁸ cells/m².

Accordingly, in some embodiments, CD30-specific CAR-expressing T cells are administered to the subject at a dose of 4 x 10⁷ cells/m², wherein the dose is split between two separate time points, wherein 2 x 10⁷ cells/m² are administered at a first time point and 2 x 10⁷ cells/m² are administered at the second time point.

In some embodiments, CD30-specific CAR-expressing T cells are administered to the subject at a dose of 1 x 10⁸ cells/m², wherein the dose is split between two separate time points, wherein 5 x 10⁷ cells/m² are administered at a first time point and 5 x 10⁷ cells/m² are administered at the second time point.

In some embodiments, CD30-specific CAR-expressing T cells are administered to the subject at a dose of 4 x 10⁸ cells/m², wherein the dose is split between two separate time points, wherein 2 x 10⁸ cells/m² are administered at a first time point and 2 x 10⁸ cells/m² are administered at the second time point.

References to CD30-specific CAR-expressing T cells being administered on day 0, day 1 , day 2, day 3, day 4, etc. are to be interpreted to mean that they are administered at any time on day 0, day 1 , day 2, day 3 or day 4.

The present disclosure provides a method of treating a CD30-positive cancer in a subject, the method comprising administering 50% of a dose of CD30-specific CAR-expressing T cells/m² on day 0 and administering 50% of a dose of CD30-specific CAR-expressing T cells on day 2, 3 or 4.

The present disclosure also provides a method of treating a CD30-positive cancer in a subject, the method comprising administering 2 x 10⁷ to 2 x 10⁸ CD30-specific CAR-expressing T cells/m² on day 0 and administering 2 x 10⁷ - 2 x 10⁸ CD30-specific CAR-expressing T cells on day 2, 3 or 4.

The present disclosure also provides a method of treating a CD30-positive cancer in a subject, the method comprising administering 2 x 10⁷ CD30-specific CAR-expressing T cells/m² on day 0 and administering 2 x 10⁷ CD30-specific CAR-expressing T cells on day 2, 3 or 4.

The present disclosure also provides a method of treating a CD30-positive cancer in a subject, the method comprising administering 5 x 10⁷ CD30-specific CAR-expressing T cells/m² on day 0 and administering 5 x 10⁷ CD30-specific CAR-expressing T cells on day 2, 3 or 4.

The present disclosure also provides a method of treating a CD30-positive cancer in a subject, the method comprising administering 2 x 10⁸ CD30-specific CAR-expressing T cells/m² on day 0 and administering 2 x 10⁸ CD30-specific CAR-expressing T cells on day 2, 3 or 4.

Multiple (e.g. 2, 3, 4 or more) doses or total doses of CD30-specific CAR-expressing T cells may be provided. Multiple doses may be separated by a predetermined time interval, which may be selected to be one of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, or more hours or 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days, or 1 , 2, 3, 4, 5, or 6 months. The decision to administer one or more further dose(s) of CD30- specific CAR-expressing T cells may be made based on the response of the subject to treatment, and/or availability of CD30-specific CAR-expressing T cells.

In some embodiments, the methods of the present disclosure may comprise further therapeutic or prophylactic intervention, e.g. additional chemotherapy, immunotherapy, radiotherapy, surgery, vaccination and/or hormone therapy. Such further therapeutic or prophylactic intervention may occur before, during and/or after the administration of lymphodepleting chemotherapy or CD30-specific CAR- expressing T cells in accordance with the methods of the present disclosure, and may occur the same or different routes of administration.

Additional chemotherapy may employ a chemical entity, e.g. small molecule pharmaceutical, antibiotic, DNA intercalator, protein inhibitor (e.g. kinase inhibitor), or a biological agent, e.g. antibody, antibody fragment, aptamer, nucleic acid (e.g. DNA, RNA), peptide, polypeptide, or protein. The drug may be formulated as a pharmaceutical composition or medicament. The formulation may comprise one or more drugs (e.g. one or more active agents) together with one or more pharmaceutically acceptable diluents, excipients or carriers. Radiotherapy may employ ionising radiation, e.g. radiotherapy using X-rays or y- rays.

Prior to administration of a lymphodepleting chemotherapy and/or CD30-specific chimeric antigen receptor (CAR)-expressing T cells in accordance with aspects of the present disclosure, a subject may be administered bridging therapy. Bridging therapy may be administered to the subject after blood sample collection, and prior to administration of a lymphodepleting chemotherapy. Bridging therapy is therapy designed to carry the subject through to treatment in accordance with the methods of the present disclosure. The decision to administer bridging therapy at the discretion and under the control of medical practitioners. Bridging therapy may comprise administering or more of steroids, chemotherapy, palliative radiation therapy, an immune checkpoint inhibitor or anti-CD30 antibodies to the subject.

Bridging therapy may be followed by a washout period prior to administration of a lymphodepleting chemotherapy in accordance with the methods of the present disclosure. The washout period ensures adequate recovery from toxicity associated with the bridging therapy prior to administration of the first dose of a lymphodepleting chemotherapeutic agent in accordance with a lymphodepleting chemotherapy according to the present disclosure. The appropriate washout period depends on the particular bridging therapy employed. Where steroids are administered as a bridging therapy, the washout period may be 1 week. Where chemotherapy is administered as a bridging therapy, the washout period may be 3 weeks. Where palliative radiation therapy is administered as a bridging therapy, the washout period may be 2 weeks. Where an immune checkpoint inhibitor is administered as a bridging therapy, the washout period may be 3 weeks. Where anti-CD30 antibodies are administered as a bridging therapy, the washout period may be 8 weeks.

Particular exemplary embodiments of methods of treatment in accordance with the present disclosure are described below. In some embodiments, the method comprises:

(i) administering fludarabine at a dose of 30 mg/m²/day and cyclophosphamide at a dose of 500 mg/m²/day to a subject for 3 consecutive days,

(ii) 2 to 5 days (e.g. 2 days) after the final day of administration of fludarabine and cyclophosphamide, administering a first dose of CD30-specific CAR-expressing T cells to the subject at a dose of 2 x 10⁷ cells/m², and

(iii) 2 to 4 days (e.g. 3 days) after the first dose of CD30-specific CAR-expressing T cells, administering a second dose of CD30-specific CAR-expressing T cells to the subject at a dose of 2 x 10⁷ cells/m².

In some embodiments, the method comprises:

(i) administering fludarabine at a dose of 30 mg/m²/day and cyclophosphamide at a dose of 500 mg/m²/day to a subject for 3 consecutive days, and

(ii) 2 to 5 days (e.g. 2 days) after the final day of administration of fludarabine and cyclophosphamide, administering a first dose CD30-specific CAR-expressing T cells to the subject at a dose of 5 x 10⁷ cells/m².

(iii) 2 to 4 days (e.g. 3 days) after the first dose of CD30-specific CAR-expressing T cells, administering a second dose of CD30-specific CAR-expressing T cells to the subject at a dose of 5 x 10⁷ cells/m².

In some embodiments, the method comprises:

(ii) 2 to 5 days (e.g. 2 days) after the final day of administration of fludarabine and cyclophosphamide, administering a first dose of CD30-specific CAR-expressing T cells to the subject at a dose of 2 x 10⁸ cells/m².

(iii) 2 to 4 days (e.g. 3 days) after the first dose of CD30-specific CAR-expressing T cells, administering a second dose of CD30-specific CAR-expressing T cells to the subject at a dose of 2 x 10⁸ cells/m².

The subject in accordance with aspects the present disclosure may be any animal or human. The subject is preferably mammalian, more preferably human. The subject may be a non-human mammal, but is more preferably human. The subject may be a patient. The subject may be male or female. The subject may be an adult subject (aged >18 years), a pediatric subject (aged <18 years), or an adolescent subject (aged >12 and <21 years; e.g. an early adolescent (aged >12 and <14 years), middle adolescent (aged >15 and <17 years), or late adolescent (aged >18 and <21 years)). The subject may be aged <75 years.

The subject may have a CD30-positive cancer (e.g. a CD30-positive cancer according to an embodiment described herein). The subject may have a CD30-positive tumor. The subject may have been determined to have a CD30-positive cancer, may have been diagnosed with a CD30-positive cancer, may be suspected of having a CD30-positive cancer, or may be at risk of developing a CD30-positive cancer. In some embodiments, the subject may be selected for treatment in accordance with the methods of the present disclosure based on determination that the subject has a CD30-positive cancer. The subject may have at least one measurable lesions according to the Revised Criteria for Response Assessment: The Lugano Classification (described e.g. in Cheson et al., J Clin Oncol (2014) 32: 3059-3068, which is hereby incorporated by reference in its entirety).

The subject may be a subject that has relapsed following a treatment for the cancer. The subject may have responded to a treatment for the cancer (e.g. a first line therapy for the cancer), but the cancer may have subsequently re-emerged/progressed, e.g. after a period of remission.

The subject may be a subject that failed to respond to a treatment for the cancer. The subject may not have responded to a treatment for the cancer (e.g. a first line therapy for the cancer). The subject may not have displayed a partial or complete response to a treatment for the cancer (e.g. a first line therapy for the cancer).

The subject may be autogeneic/autologous with respect to the source of the cells from which the CD30- specific CAR-expressing T cells administered in accordance with the methods of the disclosure are derived. The subject to which the CD30-specific CAR-expressing T cells are administered may be the same subject from which the blood sample or cells are obtained for the production of the CD30-specific CAR-expressing T cells. The subject to which the CD30-specific CAR-expressing T cells are administered may be genetically identical to the subject from which the blood sample or cells are obtained for the production of the CD30-specific CAR-expressing T cells. The subject to which the CD30-specific CAR- expressing T cells are administered may comprise MHC/HLA genes encoding MHC/HLA molecules which are identical to the MHC/HLA molecules encoded by the MHC/HLA genes of the subject from which the blood sample or cells are obtained for the production of the CD30-specific CAR-expressing T cells.

Alternatively, the subject may be allogeneic/non-autologous with respect to the source of the cells from which the CD30-specific CAR-expressing T cells administered in accordance with the methods of the disclosure are derived. The subject to which the CD30-specific CAR-expressing T cells are administered may be a different subject to the subject from which the blood sample or cells are obtained for the production of the CD30-specific CAR-expressing T cells. The subject to which the CD30-specific CAR- expressing T cells are administered may be genetically non-identical to the subject from which the blood sample or cells are obtained for the production of the CD30-specific CAR-expressing T cells. The subject to which the CD30-specific CAR-expressing T cells are administered may comprise MHC/HLA genes encoding MHC/HLA molecules which are identical to the MHC/HLA molecules encoded by the MHC/HLA genes of the subject from which the blood sample or cells are obtained for the production of the CD30- specific CAR-expressing T cells.

A subject may be an allogeneic subject with respect to an intervention in accordance with the present disclosure. A subject to be treated/prevented in accordance with the present disclosure may be genetically non-identical to the subject from which the CAR-expressing virus-specific immune cells are derived. A subject to be treated/prevented in accordance with the present disclosure may be HLA mismatched with respect to the subject from which the CAR-expressing virus-specific immune cells are derived. A subject to be treated/prevented in accordance with the present disclosure may be HLA matched with respect to the subject from which the CAR-expressing virus-specific immune cells are derived.

The subject to which cells are administered in accordance with the present disclosure may be allogeneic/non-autologous with respect to the source from which the cells are/were derived. The subject to which cells are administered may be a different subject to the subject from which cells are/were obtained for the production of the cells to be administered. The subject to which the cells are administered may be genetically non-identical to the subject from which cells are/were obtained for the production of the cells to be administered.

Effects achieved by treatment according to the present disclosure

Methods of the present disclosure may be characterised by reference to treatment effects and/or clinical outcomes achieved by the method.

Treatment of a subject in accordance with the methods of the present disclosure achieves one or more of the following treatment effects: reduces the number of CD30-positive cancer cells in the subject, reduces the size of a CD30-positive tumor/lesion in the subject, inhibits (e.g. prevents or slows) growth of CD30- positive cancer cells in the subject, inhibits (e.g. prevents or slows) growth of a CD30-positive tumor/lesion in the subject, inhibits (e.g. prevents or slows) the development/progression of a CD30- positive cancer (e.g. to a later stage, or metastasis), reduces the severity of symptoms of a CD30-positive cancer in the subject, increases survival of the subject (e.g. progression free survival or overall survival), reduces a correlate of the number or activity of CD30-positive cancer cells in the subject, and/or reduces CD30-positive cancer burden in the subject.

Subjects may be evaluated in accordance with the Revised Criteria for Response Assessment: The Lugano Classification (described e.g. in Cheson et al., J Clin Oncol (2014) 32: 3059-3068, incorporated by reference hereinabove) in order to determine their response to treatment. In some embodiments, treatment of a subject in accordance with the methods of the present disclosure achieves one of the following: complete response, partial response, or stable disease.

Methods of the present disclosure may be characterised by reference to effects achieved/responses observed at a population level. That is, in some embodiments the methods of the present disclosure may be characterised by reference to effects achieved/responses observed when the treatment is administered to more than one subject, e.g. a population of subjects. A population of subjects may comprise 2 or more, e.g. one of 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90 or 100 or more subjects.

Effects achieved/responses observed at a population level may be expressed in terms of the proportion (e.g. percentage) of treated subjects displaying a given clinical outcome (e.g. complete response, partial response, overall response (compete response + partial response), stable disease, progressive disease). The proportion of treated subjects displaying a given clinical outcome may be referred to as the “rate” for the clinical outcome. By way of illustration, the percentage of subjects displaying a complete response to treatment may be referred to as the complete response rate.

In some embodiments, treatment in accordance with the methods of the present disclosure achieves an overall response rate (i.e. complete response plus partial response) of 50% or greater, e.g. one of 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% or greater, or an overall response rate of 100%. In some embodiments, treatment in accordance with the methods of the present disclosure achieves an overall response rate of 70% or greater, e.g. one of 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80% or 81% or greater.

In some embodiments, treatment in accordance with the methods of the present disclosure achieves a complete response rate of 50% or greater, e.g. one of 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% or greater, or a complete response rate of 100%. In some embodiments, treatment in accordance with the methods of the present disclosure achieves a complete response rate of 70% or greater, e.g. one of 71%, 72%, 73%, 74% or 75% or greater.

In some embodiments, treatment in accordance with the methods of the present disclosure achieves a progressive disease rate of 50% or less, e.g. one of 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% or 5% or less, or a progressive disease rate of 0%. In some embodiments, treatment in accordance with the methods of the present disclosure achieves a progressive disease rate of 30% or less, e.g. one of 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14% or 13% or less.

In some embodiments, treatment in accordance with the methods of the present disclosure achieves a 1 year progression free survival rate of 20% or greater, e.g. one of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or greater, or a 1 year progression free survival rate of 100%. In some embodiments, treatment in accordance with the methods of the present disclosure achieves a complete response rate of 40% or greater, e.g. one of 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56% or 57% or greater.

In some embodiments, treatment in accordance with the methods of the present disclosure achieves a median progression free survival of 1 month or greater, e.g. one of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23 or 24 months or greater. In some embodiments, treatment in accordance with the methods of the present disclosure achieves a median progression free survival of 9 months or greater, e.g. one of 10, 11 , 12 or 13 months or greater.

In some embodiments, treatment in accordance with the methods of the present disclosure achieves a 1 year overall survival rate of 90% or greater, e.g. one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater, or 1 year overall survival rate of 100%. In some embodiments, treatment in accordance with the methods of the present disclosure achieves a median overall survival of 6 months or greater, e.g. one of 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23 or 24 months or greater.

In some embodiments, treatment in accordance with the methods of the present disclosure achieves a 1 year duration of response rate (e.g. in subjects achieving a complete response or a partial response) of 20% or greater, e.g. one of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or greater, or a 1 year duration of response rate of 100%.

In some embodiments, treatment in accordance with the methods of the present disclosure achieves a median duration of response (e.g. in subjects achieving a complete response or a partial response) of 1 month or greater, e.g. one of 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23 or 24 months or greater.

In embodiments of the present disclosure, treatment effects and clinical outcomes may be characterised by reference to the effects/ outcomes (e.g. clinical responses) achieved by a treatment in accordance with a reference method. A reference method may be a method comprising administering CD30-specific CAR- expressing T cells to a subject.

In some embodiments, a reference method may comprise treatment by administering CD30-specific CAR-expressing T cells (e.g. at a dose of 2 x 10⁷ cells/m², 1 x 10⁸ cells/m² or 2 x 10⁸ cells/m²) without prior administration of a lymphodepleting chemotherapy. In some embodiments, a reference method may comprise treatment of a CD30-positive cancer by administering CD30-specific CAR-expressing T cells to a subject as described in Ramos et al., J Clin Invest. (2017) 127(9):3462-3471 , or in accordance with an intervention described for NCT01316146 (reproduced below):

Drug: CAR. CD30 T cells

Three dose levels:

Group One, 2x10^A7 cells/m^A2

Group Two, 1 x10^A8 cells/m^A2

Group Three, 2x10^A8 cells/m^A2;

Cell Administration: CAR+ activated T lymphocytes given by intravenous injection over 1 -10 minutes through either a peripheral or a central line. Expected volume = 1 -50cc.

In some embodiments, a reference method may comprise treatment by administering lymphodepleting chemotherapy comprising administering fludarabine and cyclophosphamide (e.g. at a dose of 30 mg/m²/day fludarabine and 500 mg/m²/day cyclophosphamide for three consecutive days), and subsequently (e.g. within 2 to 14 days of completion of the course of lymphodepleting chemotherapy) administering CD30-specific CAR-expressing T cells (e.g. at a dose of 2 x 10⁷ cells/m², 1 x 10⁸ cells/m² or 2 x 10⁸ cells/m²). In some embodiments, a reference method may comprise treatment of a CD30-positive cancer as described in Ramos et al., Biol Blood Marrow Transplant 25 (2019) S7-S75, Abstract 79, or in accordance with the intervention described for NCT02917083 (reproduced below):

Genetic: CAR T Cells

Three dose levels. Each patient receives one infusion of CAR modified T cells according to the following dosing schedule:

Dose Level One: 2x10^A7 cells/m². Dose Level Two: 1 x10^A8 cells/m². Dose Level Three: 2x10^A8 cells/m².

Drug: Cyclophosphamide Patients who are not recently post autologous transplant will receive three daily doses of cyclophosphamide (Cy: 500mg/m²/day) finishing at least 48 hours before T cell infusion, but no later than 2 weeks prior to infusion of the cells.

Other Name: Cytoxan

Drug: Fludarabine Patients who are not post autologous transplant will receive fludarabine (Flu: 30mg/m²/day), finishing at least 48 hours before T cell infusion, but no later than 2 weeks prior to infusion of the cells.

Treatment in accordance with the methods of the present disclosure may be associated with an improved treatment effect and/or an improved clinical outcome as compared to treatment in accordance with a reference method.

Treatment in accordance with the methods of the present disclosure may achieve one or more of: a greater reduction in the number of CD30-positive cancer cells in the subject, a greater reduction in the size of a CD30-positive tumor/lesion in the subject, greater inhibition of growth of CD30-positive cancer cells in the subject, greater inhibition of growth of a CD30-positive tumor/lesion in the subject, greater inhibition of the development/progression of a CD30-positive cancer (e.g. to a later stage, or metastasis), a greater reduction in the severity of symptoms of a CD30-positive cancer in the subject, a greater increase in survival of the subject (e.g. progression free survival or overall survival), a greater reduction in a correlate of the number or activity of CD30-positive cancer cells in the subject, and/or a greater reduction in CD30-positive cancer burden in the subject, as compared to treatment in accordance with a reference method.

A “greater” reduction/inhibition/increase may be a reduction/inhibition/increase which is greater than 1 times, e.g. one of >1 .01 times, >1 .02 times, >1 .03 times, >1 .04 times, >1 .05 times, >1 .1 times, >1 .2 times, >1 .3 times, >1 .4 times, >1 .5 times, >1 .6 times, >1 .7 times, >1 .8 times, >1 .9 times, >2 times, >3 times, >4 times, >5 times, >6 times, >7 times, >8 times, >9 times or >10 times the level of reduction/inhibition/increase achieved by the treatment in accordance with a reference method.

Reduction/inhibition may be to a level which is less than 1 times, e.g. <0.99 times, <0.95 times, <0.9 times, <0.85 times, <0.8 times, <0.75 times, <0.7 times, <0.65 times, <0.6 times, <0.55 times, <0.5 times, <0.45 times, <0.4 times, <0.35 times, <0.3 times, <0.25 times, <0.2 times, <0.15 times, <0.1 times, <0.05 times, or <0.01 times the level achieved by treatment in accordance with a reference method.

An increase may be to a level which is greater than 1 times, e.g. one of >1 .01 times, >1 .02 times, >1 .03 times, >1 .04 times, >1 .05 times, >1.1 times, >1 .2 times, >1 .3 times, >1 .4 times, >1 .5 times, >1 .6 times, >1 .7 times, >1 .8 times, >1 .9 times, >2 times, >3 times, >4 times, >5 times, >6 times, >7 times, >8 times, >9 times or >10 times the level achieved by treatment in accordance with a reference method.

In some embodiments, treatment in accordance with the methods of the present disclosure is associated with an improved clinical outcome (e.g. clinical response) as compared to the treatment in accordance with a reference method.

Treatment in accordance with the methods of the present disclosure may achieve one or more of: an increased overall response (i.e. complete response plus partial response) rate, an increased complete response rate, a reduced progressive disease rate, an increased 1 year progression free survival rate, an increased median progression free survival, an increased 1 year overall survival rate, increased median overall survival, an increased 1 year duration of response rate or, increased an increased median duration of response, as compared to the treatment in accordance with a reference method.

An “increased” rate/median may be a rate/median which is greater than 1 times, e.g. one of >1 .01 times, >1 .02 times, >1 .03 times, >1 .04 times, >1 .05 times, >1 .1 times, >1 .2 times, >1 .3 times, >1 .4 times, >1 .5 times, >1 .6 times, >1 .7 times, >1 .8 times, >1 .9 times, >2 times, >3 times, >4 times, >5 times, >6 times, >7 times, >8 times, >9 times or >10 times the rate/median achieved by the treatment in accordance with a reference method.

An “reduced” rate may be a rate which is less than 1 times, e.g. <0.99 times, <0.95 times, <0.9 times, <0.85 times, <0.8 times, <0.75 times, <0.7 times, <0.65 times, <0.6 times, <0.55 times, <0.5 times, <0.45 times, <0.4 times, <0.35 times, <0.3 times, <0.25 times, <0.2 times, <0.15 times, <0.1 times, <0.05 times, or <0.01 times the rate achieved by the treatment in accordance with a reference method.

Treatment in accordance with the methods of the present disclosure may be associated with a reduced proportion of subjects displaying adverse events, as compared to treatment in accordance with a reference method. Treatment in accordance with the methods of the present disclosure may be associated with a reduced proportion of subjects displaying one or more of the following, as compared to treatment in accordance with a reference method: lymphopenia, leukopenia, neutropenia, thrombocytopenia, anemia, hypoalbuminemia, hyponatremia, dyspnea, rash, headache, pharyngitis, lung Infection, cytokine Release 5 syndrome, grade 3/4 neutropenia at day 28, grade 3/4 thrombocytopenia at day 28, grade 3/4 anemia at day 28, prolonged grade 3/4 neutropenia (e.g. at month 3), prolonged grade 3/4 thrombocytopenia (e.g. at month 3), or prolonged grade 3/4 anemia (e.g. at month 3).

Sequence identity

Pairwise and multiple sequence alignment for the purposes of determining percent identity between two 0 or more amino acid or nucleic acid sequences can be achieved in various ways known to a person of skill in the art, for instance, using publicly available computer software such as ClustalOmega (Soding, J. 2005, Bioinformatics 21 , 951 -960), T-coffee (Notredame et al. 2000, J. Mol. Biol. (2000) 302, 205-217), Kalign (Lassmann and Sonnhammer 2005, BMC Bioinformatics, 6(298)) and MAFFT (Katoh and Standley 2013, Molecular Biology and Evolution, 30(4) 772-780 software. When using such software, the default 5 parameters, e.g. for gap penalty and extension penalty, are preferably used.

Sequences

Examples

In the following Examples, the inventors describe the generation of CD30.CAR-EBVSTs, the kinetics of CD30 expression in T cells, and the treatment of CD30-positive cancer using methods employing lymphodepleting chemotherapy and adoptive transfer of CD30-specific CAR-expressing T cells.

EXAMPLE 1 - Generation of retroviruses encoding CAR constructs

Retrovirus encoding the CD30.CAR construct was prepared by cloning cDNA encoding the CAR into the pSFG-TGFbDNRII retroviral backbone (ATUM, Newark, CA).

The plasmid carrying the CD30.CAR sequence, pSFG_CD30CAR, was transfected into HEK 293 Vec- RD114 cells using polyethylenimine (PEI). Cell culture supernatant from the transfected cells was then used to transduce HEK 293Vec-Galv cells (BioVec Pharma, Quebec, Canada) at a density of 5 x 10⁵ cells/well of a 6-well plate.

The 293Vec-Galv_CD30-CAR cells were trypsinized, and the cells were resuspended in a 15 ml tube at a concentration of 2 x 10⁶ cells/ml. Two series of dilutions were made, and 1 .65 ml of the final cell suspension was diluted and mixed with 220 ml of DMEM + 10% FCS. Two hundred pl of this suspension was transferred to wells of a 96-well plate, resulting in 30 cells per plate. The best performing clone was then selected and used to generate retrovirus-containing supernatant. The retrovirus-containing supernatant was subsequently collected, filtered and stored at -80°C until use. EXAMPLE 2 - Production of CD30.CAR-EBVSTs from donor

CD30.CAR EBVSTs were manufactured in a GMP facility. Approximately 250 to 400 mL of blood was collected from seven healthy, blood-bank approved donors after obtaining informed consent and in accordance with the guidelines established by the Declaration of Helsinki.

Peripheral blood mononuclear cells (PBMCs) were isolated from blood by density gradient centrifugation. PBMCs were depleted of CD45RA-expressing cells by magnetic cell separation using a clinical grade anti-CD45RA antibody conjugated to magnetic beads, and using and Miltenyi depletion columns (Miltenyi Biotec, Bergisch Gladbach, Germany).

1 .5-2.5 x 10⁷ PBMCs depleted of CD45RA-postive cells were seeded in 30 ml culture medium containing 47.5% Advanced RPMI, 47.5% Click’s (EHAA) medium (Irvine Scientific), 2 mM L-glutamine (Thermo Fisher Scientific) and 5% Human Platelet Lysate (HPL; Sexton Biotechnologies), supplemented with IL-7 (10 ng/ml) and IL-15 (10 ng/ml) in G-Rex10 vessels, and activated by stimulation with overlapping peptide libraries (pepmixes) comprising 15mer amino acids overlapping by 11 amino acids, and spanning the entire protein sequences of the relevant antigens. Pepmixes corresponding to EBNA1 , LMP1 , LMP2, BARF1 , BZLF1 , BRLF1 , BMLF1 , BMRF1 , BMRF2, BALF2, BNLF2a and BNLF2b were obtained from JPT Technologies (Berlin, Germany). Stimulations employed 5 ng of pepmix for each antigen per 1 x 10⁶ cells to be stimulated (i.e. for stimulations performed using 2 x 10⁷ PBMCs depleted of CD45RA-postive cells, 100 ng of each pepmix was used). Stimulation cultures were maintained at 37°C in a 5% CO2 atmosphere.

After 4-6 days, EBVSTs produced by the stimulation cultures described in the preceding paragraph were transduced with CAR-encoding retrovirus from Example 1 , as follows. 2 ml of retrovirus-containing supernatant was mixed with 150 pg Vectofusin-1 in a volume of 2 ml, giving a final volume of 4 ml, and incubated at room temperature for 5-30 min. The retrovirus:Vectofusin-1 mixture was then added to 7-10 x 10⁶ cells in 8.5 ml culture medium (described in the preceding paragraph), in T75 vessels. Cultures were maintained at 37°C in a 5% CO2 atmosphere.

Between days 8 and 10 of culture, 1 -2 x 10⁷CD30.CAR EBVSTs CD30.CAR EBVSTs produced by transduction as described in the preceding paragraph were transferred to G-Rex100 vessels, and restimulated by co-culture with irradiated (at 100 gray) ULCLs (described in Example 2), at a ratio of CD30.CAR EBVSTs to irradiated ULCLs ranging from 1 :2 to 1 :5 (typically around 1 :3). ULCLs express EBV antigens and CD30, as well as other costimulatory molecules, and therefore provide CD30.CAR EBVSTs with antigen stimulation and costimulation, inducing robust proliferation of CD30.CAR EBVSTs without loss of EBV specificity.

Re-stimulation cultures were established in 200 ml culture medium (described in paragraph 3 of section 5.1 ), and additional culture medium was added as required. 7 to 12 days later, CD30.CAR EBVSTs were harvested and cryopreserved for subsequent infusion. EXAMPLE 3 - Kinetics of CD30

The inventors investigated the kinetics of CD30 expression in host T cells.

Alloreactive host T cells from donor A, B and C were generated by 1 , 2 or 3 priming with allogeneic irradiated polymorphonuclear cells from mismatched donors. To evaluate if repeated encounters of allogeneic mismatched graft PBMCs (known as priming) affect CD30 expression on host T cells, host T cells that remained after the second or third prime were subsequently co-cultured with graft PBMCs from the same donor used for priming. CD30 expression on alloreactive host T cells was evaluated at 24, 48 and 72 hours of co-culture. (n=3 donor pairs) (Figure 1 ).

Figure 2A shows CD30 expression in host T cells across 72 hours in the absence of priming. Host T cells that were not primed were co-cultured with their respective mismatched graft PBMCs. Low levels CD30 was observed to be expressed in CD4 and CD8 T cells. Figure 2B shows CD30 expression in alloreactive host T cells that underwent 2 rounds of priming. The host T cells were co-cultured with their respective mismatched graft PBMCs. CD30 expression was observed to be up-regulated in both host CD4 and CD8 T cell compared to their un-primed counterparts. Figure 2C shows CD30 expression in alloreactive host T cells that underwent 3 rounds of priming. The host T cells were co-cultured with their respective mismatched graft PBMCs. CD30 expression was observed to be highly expressed in both host CD4 and CD8 T cells compared to their un-primed counterparts. The experiment was performed with 3 donor pairs. Altogether, the data demonstrates that repeated exposure to allogeneic mismatched graft PBMCs induces upregulation of CD30 expression in host T cells. In the absence of such repeated exposure to allogeneic mismatched graft PBMCs, CD30 expression on host T cells remains low.

The apparent slow upregulation of CD30 on alloreactive T-cells within PBMC after the first stimulation can be explained by the fact that the frequency of alloreactive T-cells within PBMC is very low (median less than 0.005%) (https://onlinelibrary.wiley.eo /do:/10.1002/e .201 46826) and cannot be detected by flow cytometry. However, as the alloreactive T-cells proliferate and reach a higher frequency within the population, they can be detected. Hence, CD30 cannot be detected until sufficient alloreactive T-cells have proliferated to be detectable with our assay (-0.1 %). For this reason, the kinetics of CD30 expression when all the T-cells in PBMCs are stimulated, using CD3 and CD28 antibodies, were analysed. This strategy showed that CD30 was upregulated within 2 days of stimulation in both CD4+ and CD8+ T-cells.

Figures 3 and 4 show CD30 expression on activated T cells. Figure 3: Non-tissue culture treated 24 well plates were coated with CD3 and CD28 antibodies overnight. Coated wells were washed with PBS and PBMCs were cultured at 1 .0 x 10⁶ cells per well in RPMI 1640 medium supplemented with 10% FBS and 1% GlutaMAX. Cells were harvested for CD30 analysis on the days indicated (N=3). Figure 4: CD4+ and CD8+ T-cells were sorted from PBMC, then 1 .0 x 10⁵ CD4+ or CD8+ T-cells were co-cultured with either 5.0 x 10³ irradiated allogeneic lymphoblastoid cell lines (LCL), 5.0 x 103 irradiated HLA-negative ULCL or were left unstimulated. Cells were cultured in 96-well round bottom plates in RPMI 1640 medium supplemented with 10% FBS and 1 % GlutaMAX. Cells were harvested for CD30 analysis on the days indicated (N=3). The results show that CD30 is not expressed on T-cells until several days after activation. The expression of CD30 is more delayed on alloreactive T cells compared to T cells broadly activated with antibodies to CD3 and CD28.

Taken together, the results provide the scientific rationale that the first dose of CD30.CAR T cells will activate alloreactive T cells which will express CD30 by day 3. On that day, a second dose of CD30.CAR T-cells will be infused and will be able to eliminate the alloreactive T cells that now express CD30 after activation by the first dose. CD30.CAR EBVSTs will be infused as a split dose with the second dose being given several (e.g. 2-4) days after the first dose. The first dose will activate alloreactive T-cells from the patient, which based on the CD30 kinetics results should express CD30 by day 2 to 3. The second dose of CD30.CAR EBVSTs that will be infused around day 3 should encounter CD30+ alloreactive T-cells and eliminate them.

Study rationale:

The purpose of this protocol is to study the safety and antitumor activity of allogeneic T cells directed to the CD30 antigen by means of a chimeric antigen receptor (CAR). EBV specific T cells (EBVSTs) are used as the allogeneic cell platform. These cells are virus-specific rather than alloantigen reactive and have been shown to produce little or no graft-versus-host disease (GvHD) in clinical studies (NCT01316146; Ramos et al., J Clin Invest. (2017) 127(9):3462-3471 ; NCT02917083; Ramos et al., Biol Blood Marrow Transplant 25 (2019) S7-S75, Abstract 79). Because they express a CD30 CAR, their activity against CD30+ tumors should be coupled with resistant to host-versus-graft rejection (HvG), since allo-reactive host T cells express CD30 as a result of allo-activation.

Objectives and endpoints:

Primary objectives:

To evaluate the safety of one dose of allogeneic CD30 Chimeric Antigen Receptor Epstein Barr Virus- Specific T Lymphocytes (CD30.CAR-EBVSTs) in patients with CD30+ refractory/relapsed Hodgkin Lymphoma (HL) or non-Hodgkin Lymphoma (NHL) after lymphodepleting chemotherapy.

Secondary objectives:

To measure the preliminary antitumor effect of allogeneic CD30.CAR-EBVST cells in this patient population, including

• Objective response (OR) rate (ORR)

• Duration of response (DR)

• Stable disease (SD) rate

• Duration of SD

• Progression free survival (PFS) Study Population:

The study population includes adults and pediatric patients aged 12 to 75 years with relapsed or refractory CD30 positive Hodgkin Lymphoma, non-Hodgkin-Lymphoma, ALK-negative anaplastic T cell lymphoma or other peripheral T-cell lymphoma, or ALK-positive anaplastic T cell lymphoma.

Patients may have previously received an autologous and/or allogeneic stem cell transplant.

Study Design:

This is a Phase I dose-escalation trial, designed to evaluate the safety of allogeneic CD30.CAR-EBVSTs in patients with CD30+ refractory/relapsed HL or NHL after lymphodepleting chemotherapy.

Study treatment:

Lymphodepletion chemotherapy: Patients will receive three daily doses of cyclophosphamide (Cy: 500 mg/m²/day) together with fludarabine (Flu: 30 mg/m²/day), finishing at least 48 hours before the first T cell infusion, but no later than 2 weeks prior to infusion of the cells. Infusions will be given following hospital/pharmacy recommendations. However, at a minimum, the cyclophosphamide should be infused over 1 hour and the fludarabine should be infused over 30 minutes.

CD30.CAR-T cell infusion: Each patient will receive a total dose of CAR modified T cells (split between 2 infusions 2-4 days apart) according to the following dosing schedule:

• Dose Level 1 : 4 x 10⁷ CD30.CAR-EBVST cells

• Dose Level 2: 1 x 10⁸ CD30.CAR-EBVST cells

• Dose Level 3: 4 x 10⁸ CD30.CAR-EBVST cells

The CD30.CAR-EBVST cells will be infused into the vein through an IV line at half the assigned dose with each infusion.

***

The features disclosed in the foregoing description, or in the following claims, or in the accompanying drawings, expressed in their specific forms or in terms of a means for performing the disclosed function, or a method or process for obtaining the disclosed results, as appropriate, may, separately, or in any combination of such features, be utilised for realising the invention in diverse forms thereof.

While the invention has been described in conjunction with the exemplary embodiments described above, many equivalent modifications and variations will be apparent to those skilled in the art when given this disclosure. Accordingly, the exemplary embodiments of the invention set forth above are considered to be illustrative and not limiting. Various changes to the described embodiments may be made without departing from the spirit and scope of the invention. For the avoidance of any doubt, any theoretical explanations provided herein are provided for the purposes of improving the understanding of a reader. The inventors do not wish to be bound by any of these theoretical explanations.

Any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

Throughout this specification, including the claims which follow, unless the context requires otherwise, the word “comprise” and “include”, and variations such as “comprises”, “comprising”, and “including” will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent “about,” it will be understood that the particular value forms another embodiment. The term “about” in relation to a numerical value is optional and means for example +/- 10%.

Claims

Claims:

1 . A method of treating a CD30-positive cancer in a subject, comprising administering a dose of CD30-specific chimeric antigen receptor (CAR)-expressing T cells to the subject, wherein the dose is administered in two parts at two time points, wherein a first part of the dose is administered at a first time point and the remaining part of the dose is administered at a second time point, wherein the first and second time points are 2 to 4 days apart.

2. A composition comprising CD30-specific chimeric antigen receptor (CAR)-expressing T cells for use in a method of treating a CD30-positive cancer, wherein the method comprises administering a dose of CD30-specific chimeric antigen receptor (CAR)-expressing T cells to the subject, wherein the dose is administered in two parts at two time points, wherein a first part of the dose is administered at a first time point and the remaining part of the dose is administered at a second time point, wherein the first and second time points are 2 to 4 days apart.

3. Use of a composition comprising CD30-specific chimeric antigen receptor (CAR)-expressing T cells in the manufacture of a medicament for use in a method of treating a CD30-positive cancer, wherein the method comprises administering a dose of CD30-specific chimeric antigen receptor (CAR)-expressing T cells to the subject, wherein the dose is administered in two parts at two time points, wherein a first part of the dose is administered at a first time point and the remaining part of the dose is administered at a second time point, wherein the first and second time points are 2 to 4 days apart.

4. The method, the composition for use, or the use according to any one of claims 1 to 3, wherein the CD30-specific chimeric antigen receptor (CAR)-expressing T cells are allogeneic to the subject.

5. A method of eliminating alloreactive T cells in a subject with a CD30-positive cancer, comprising administering a dose of allogeneic CD30-specific chimeric antigen receptor (CAR)-expressing T cells to the subject, wherein a first part of the dose is administered at a first time point and the remaining part of the dose is administered at a second time point, wherein the first and second time points are 2 to 4 days apart.

6. A composition comprising allogeneic CD30-specific chimeric antigen receptor (CAR)-expressing T cells for use in a method of eliminating alloreactive T cells in a subject with a CD30-positive cancer, wherein the method comprises administering a dose of CD30-specific chimeric antigen receptor (CAR)-expressing T cells to the subject, wherein the dose is administered in two parts at two time points, wherein a first part of the dose is administered at a first time point and the remaining part of the dose is administered at a second time point, wherein the first and second time points are 2 to 4 days apart. Use of a composition comprising allogeneic CD30-specific chimeric antigen receptor (CAR)- expressing T cells in the manufacture of a medicament for use in a method of eliminating alloreactive T cells in a subject with a CD30-positive cancer, wherein the method comprises administering a dose of CD30-specific chimeric antigen receptor (CAR)-expressing T cells to the subject, wherein the dose is administered in two parts at two time points, wherein a first part of the dose is administered at a first time point and the remaining part of the dose is administered at a second time point, wherein the first and second time points are 2 to 4 days apart. The method, the composition for use, or the use according to any one of claims 4 to 7, wherein the method comprises adoptive transfer of allogeneic CD30-specific chimeric antigen receptor (CAR)-expressing T cells. The method, the composition for use, or the use according to any one of claims 1 to 8, wherein the CD30-specific chimeric antigen receptor (CAR)-expressing T cells are virus-specific T cells. The method, the composition for use, or the use according to claim 9, wherein the virus-specific T cells are specific for Epstein-Barr virus (EBV). The method, the composition for use, or the use according to any one of claims 1 to 9, wherein the first and second time points are 3 days apart. The method, the composition for use, or the use according to any one of claims 1 to 10, wherein 50% of the dose is administered at the first time point, and 50% of the dose is administered at the second time point. The method, the composition for use, or the use according to any one of claims 1 to 11 , wherein 50% of the dose is administered on day 0, and 50% of the dose is administered on day 3. The method, the composition for use, or the use according to any one of claims 1 to 12, wherein the dose is about 4 x 10⁷ to about 4 x 10⁸ CD30-specific CAR-expressing T cells/m² to the subject. The method, the composition for use, or the use according to any one of claims 1 to 13, wherein the dose is about 4 x 10⁷ CD30-specific CAR-expressing T cells/m². The method, the composition for use, or the use according to any one of claims 1 to 13, wherein the dose is about 1 x 10⁸ CD30-specific CAR-expressing T cells/m². The method, the composition for use, or the use according to any one of claims 1 to 13, wherein the dose is about 4 x 10⁸ CD30-specific CAR-expressing T cells/m².

18. A method of treating a CD30-positive cancer in a subject, comprising administering CD30-specific chimeric antigen receptor (CAR)-expressing T cells to the subject, wherein the method comprises administering a first dose of the CD30-specific chimeric antigen receptor (CAR)-expressing T cells to the subject, and subsequently administering a second dose of the CD30-specific chimeric antigen receptor (CAR)-expressing T cells to the subject, wherein the first and second doses are administered 2 to 4 days apart.

19. A composition comprising CD30-specific chimeric antigen receptor (CAR)-expressing T cells for use in a method of treating a CD30-positive cancer, wherein the method comprises administering a first dose of the CD30-specific chimeric antigen receptor (CAR)-expressing T cells to the subject, and subsequently administering a second dose of the CD30-specific chimeric antigen receptor (CAR)-expressing T cells to the subject, wherein the first and second doses are administered 2 to 4 days apart.

20. Use of a composition comprising CD30-specific chimeric antigen receptor (CAR)-expressing T cells in the manufacture of a medicament for use in a method of treating a CD30-positive cancer, wherein the method comprises administering a first dose of the CD30-specific chimeric antigen receptor (CAR)-expressing T cells to the subject, and subsequently administering a second dose of the CD30-specific chimeric antigen receptor (CAR)-expressing T cells to the subject, wherein the first and second doses are administered 2 to 4 days apart.

21 . The method, the composition for use, or the use according to any one of claims 18 to 20, wherein the CD30-specific chimeric antigen receptor (CAR)-expressing T cells are allogeneic to the subject.

22. The method, the composition for use, or the use according to any one of claims 18 to 21 , wherein the method comprises adoptive transfer of allogeneic CD30-specific chimeric antigen receptor (CAR)-expressing T cells.

23. The method, the composition for use, or the use according to any one of claims 18 to 22, wherein CD30-specific chimeric antigen receptor (CAR)-expressing T cells are virus-specific T cells.

24. The method, the composition for use, or the use according to claim 23, wherein the virus-specific T cells are specific for Epstein-Barr virus.

25. The method, the composition for use, or the use according to any one of claims 18 to 24, wherein the first and second doses are administered 3 days apart.

26. The method, the composition for use, or the use according to any one of claims 18 to 25, wherein the first dose is administered on day 0 and the second dose is administered on day 3.

27. The method, the composition for use, or the use according to any one of claims 18 to 26, wherein the first dose is about 2 x 10⁷ to about 2 x 10⁸ CD30-specific CAR-expressing T cells/m².

28. The method, the composition for use, or the use according to any one of claims 18 to 27, wherein the second dose is about 2 x 10⁷ to about 2 x 10⁸ CD30-specific CAR-expressing T cells/m².

29. The method, the composition for use, or the use according to any one of claims 18 to 28, wherein the total dose comprising the first and second dose is about 4 x 10⁷ to about 4 x 10⁸ CD30- specific CAR-expressing T cells/m².

30. The method, the composition for use, or the use according to any one of claims 18 to 29, wherein the total dose comprising the first and second dose is about 4 x 10⁷ CD30-specific CAR- expressing T cells/m².

31 . The method, the composition for use, or the use according to any one of claims 18 to 30, wherein the total dose comprising the first and second dose is about 1 x 10⁸ CD30-specific CAR- expressing T cells/m².

32. The method, the composition for use, or the use according to any one of claims 18 to 31 , wherein the total dose comprising the first and second dose is about 4 x 10⁸ CD30-specific CAR- expressing T cells/m².

33. A method of treating a CD30-positive cancer in a subject, comprising administering a CD30- specific chimeric antigen receptor (CAR)-expressing T cells to the subject, wherein the method comprises administering a total dose of about 4 x 10⁷ to about 4 x 10⁸ CD30-specific CAR- expressing T cells/m² to the subject, wherein the total dose comprises a first dose and a second dose, wherein the first and second dose are administered 2 to 4 days apart.

34. A composition comprising CD30-specific chimeric antigen receptor (CAR)-expressing T cells for use in a method of treating a CD30-positive cancer in a subject, wherein the method comprises administering CD30-specific chimeric antigen receptor (CAR)-expressing T cells to the subject, wherein the method comprises administering a total dose of about 4 x 10⁷ to about 4 x 10⁸ CD30- specific CAR-expressing T cells/m² to the subject, wherein the total dose comprises a first dose and a second dose, wherein the first and second dose are administered 2 to 4 days apart.

35. Use of a composition comprising CD30-specific chimeric antigen receptor (CAR)-expressing T cells in the manufacture of a medicament for use in a method of treating a CD30-positive cancer in a subject, wherein the method comprises administering CD30-specific chimeric antigen receptor (CAR)-expressing T cells to the subject, wherein the method comprises administering a total dose of about 4 x 10⁷ to about 4 x 10⁸ CD30-specific CAR-expressing T cells/m² to the subject, wherein the total dose comprises a first dose and a second dose, wherein the first and second dose are administered 2 to 4 days apart.

36. The method, the composition for use, or the use according to any one of claims 33 to 35, wherein the CD30-specific chimeric antigen receptor (CAR)-expressing T cells are allogeneic to the subject.

37. The method, the composition for use, or the use according to any one of claims 33 to 36, wherein the method comprises adoptive transfer of allogeneic CD30-specific chimeric antigen receptor (CAR)-expressing T cells.

38. The method, the composition for use, or the use according to any one of claims 33 to 37, wherein the CD30-specific chimeric antigen receptor (CAR)-expressing T cells are virus-specific T cells.

39. The method, the composition for use, or the use according to claim 38, wherein the virus-specific T cells are specific for Epstein-Barr virus (EBV).

40. The method, the composition for use, or the use according to any one of claims 33 to 39, wherein the first and second dose are administered 3 days apart.

41 . The method, the composition for use, or the use according to any one of claims 33 to 40, wherein the first dose is 50% of the total dose, and the second dose is 50% of the total dose.

42. The method, the composition for use, or the use according to any one of claims 33 to 41 , wherein the first and second doses are about 2 x 10⁷ to about 2 x 10⁸ CD30-specific CAR-expressing T cells/m².

43. The method, the composition for use, or the use according to any one of claims 33 to 42, wherein the total dose is about 4 x 10⁷ to about 4 x 10⁸ CD30-specific CAR-expressing T cells/m².

44. The method, the composition for use, or the use according to any one of claims 33 to 43, wherein the total dose is about 4 x 10⁷ CD30-specific CAR-expressing T cells/m².

45. The method, the composition for use, or the use according to any one of claims 33 to 44, wherein the total dose is about 1 x 10⁸ CD30-specific CAR-expressing T cells/m².

46. The method, the composition for use, or the use according to any one of claims 33 to 45, wherein the total dose is about 4 x 10⁸ CD30-specific CAR-expressing T cells/m².

47. The method, the composition for use, or the use according to any one of claims 1 to 46, wherein prior to administration of the CD30-specific chimeric antigen receptor (CAR)-expressing T cells, a lymphodepleting chemotherapy is administered to the subject.

48. The method, the composition for use, or the use according to claim 47, wherein the lymphodepleting chemotherapy comprises fludarabine and cyclophosphamide.

49. The method, the composition for use, or the use according to claim 47 or 48, wherein fludarabine is administered at a dose of 15 to 60 mg/m² per day, for 2 to 6 consecutive days.

50. The method, the composition for use or the use according to any one of claims 47 to 49, wherein fludarabine is administered at a dose of 30 mg/m² per day, for 3 consecutive days.

51 . The method, the composition for use, or the use according to any one of claims 47 to 50, wherein cyclophosphamide is administered at a dose of 250 to 1000 mg/m² per day, for 2 to 6 consecutive days.

52. The method, the composition for use, or the use according to any one of claims 47 to 51 , wherein cyclophosphamide is administered at a dose of 500 mg/m² per day, for 3 consecutive days.

53. The method, the composition for use, or the use according to any one of claims 47 to 52, wherein fludarabine is administered at a dose of 30 mg/m² per day and cyclophosphamide is administered at a dose of 500 mg/m² per day to a subject for 3 consecutive days.

54. The method, the composition for use, or the use according to claim 47, wherein the lymphodepleting chemotherapy comprises cyclophosphamide and bendamustine.

55. The method, the composition for use, or the use according to claim 54, wherein cyclophosphamide is administered at a dose of 250 to 1000 mg/m² per day, for 2 to 6 consecutive days.

56. The method, the composition for use, or the use according to claim 54 or 55, wherein cyclophosphamide is administered at a dose of 500 mg/m² per day, for 3 consecutive days.

57. The method, the composition for use, or the use according to any one of claims 54 to 56, wherein bendamustine is administered at a dose of 35 to 140 mg/m² per day, for 2 to 6 consecutive days.

58. The method, the composition for use, or the use according to any one of claims 54 to 57, wherein bendamustine is administered at a dose of 70 mg/m² per day, for 3 consecutive days.

59. The method, the composition for use, or the use according to any one of claims 54 to 58, wherein cyclophosphamide is administered at a dose of 500 mg/m² per day and bendamustine is administered at a dose of 70 mg/m² per day to a subject for 3 consecutive days.

60. The method, the composition for use, or the use according to any one of claims 1 to 59, wherein the CD30-positive cancer is selected from: a hematological cancer, a solid cancer, a hematopoietic malignancy, Hodgkin’s lymphoma, anaplastic large cell lymphoma, peripheral T cell lymphoma, peripheral T cell lymphoma not otherwise specified, T cell leukemia, T cell lymphoma, cutaneous T cell lymphoma, NK-T cell lymphoma, extranodal NK-T cell lymphoma, non-Hodgkin’s lymphoma, B cell non-Hodgkin’s lymphoma, diffuse large B cell lymphoma, diffuse large B cell lymphoma not otherwise specified, EBV-positive B cell lymphoma, EBV-positive diffuse large B cell lymphoma, primary mediastinal B cell lymphoma, advanced systemic mastocytosis, a germ cell tumor and testicular embryonal carcinoma.

61 . The method, the composition for use or the use according to any one of claims 1 to 60, wherein the CD30-positive cancer is selected from: Hodgkin’s lymphoma, non-Hodgkin’s lymphoma, anaplastic large cell lymphoma, peripheral T cell lymphoma not otherwise specified, extranodal NK-T cell lymphoma, diffuse large B cell lymphoma not otherwise specified and primary mediastinal large B-cell lymphoma.

62. The method, the composition for use or the use according to any one of claims 1 to 61 , wherein the subject has previously failed therapy for the CD30-positive cancer.

63. The method, the composition for use or the use according to any one of claims 1 to 62, wherein the CD30-positive cancer is a relapsed or refractory CD30-positive cancer.

64. The method, the composition for use or the use according to any one of claims 1 to 63, wherein the CD30-specific CAR-expressing T cells comprise a CAR comprising: (i) an antigen-binding domain which binds specifically to CD30, (ii) a transmembrane domain, and (iii) a signalling domain, wherein the signalling domain comprises: (a) an amino acid sequence derived from the intracellular domain of CD28, and (b) an amino acid sequence comprising an immunoreceptor tyrosine-based activation motif (ITAM).

65. The method, the composition for use or the use according to claim 64, wherein the signalling domain comprises an amino acid sequence having at least 80% amino acid sequence identity to SEQ ID NO:26.

66. The method, the composition for use or the use according to claim 64 or 65, wherein the transmembrane domain is derived from the transmembrane domain of CD28.

67. The method, the composition for use or the use according to any one of claims 64 to 66, wherein the transmembrane domain comprises an amino acid sequence having at least 80% amino acid sequence identity to SEQ ID NO:20.

68. The method, the composition for use or the use according to any one of claims 64 to 67, wherein the antigen-binding domain comprises an amino acid sequence having at least 80% amino acid sequence identity to SEQ ID NO:14, and an amino acid sequence having at least 80% amino acid sequence identity to SEQ ID NO:15.

69. The method, the composition for use or the use according to any one of claims 64 to 68, wherein the antigen-binding domain comprises an amino acid sequence having at least 80% amino acid sequence identity to SEQ ID NO:18.

70. The method, the composition for use or the use according to any one of claims 64 to 69, wherein the signalling domain comprises: (a) an amino acid sequence derived from the intracellular domain of CD3£.

71 . The method, the composition for use or the use according to any one of claims 64 to 70, wherein the signalling domain comprises an amino acid sequence having at least 80% amino acid sequence identity to SEQ ID NO:25.

72. The method, the composition for use or the use according to any one of claims 64 to 71 , wherein the CAR additionally comprises a hinge region provided between the antigen-binding domain and the transmembrane domain.

73. The method, the composition for use or the use according to claim 72, wherein the hinge region comprises an amino acid sequence having at least 80% amino acid sequence identity to SEQ ID NO:33.

74. The method, the composition for use or the use according to any one of claims 64 to 73, wherein the CAR comprises an amino acid sequence having at least 80% amino acid sequence identity to SEQ ID NO:35 or 36.