WO2023104910A1

WO2023104910A1 - Treatment of lymphoma

Info

Publication number: WO2023104910A1
Application number: PCT/EP2022/084842
Authority: WO
Inventors: Ivan David Horak; Aung MYO
Original assignee: Tessa Therapeutics Ltd.; Bristol-Myers Squibb Company; CLEGG, Richard Ian
Priority date: 2021-12-08
Filing date: 2022-12-07
Publication date: 2023-06-15
Also published as: TW202328438A

Abstract

There is provided a treatment for patients with CD30+ cancers, and particularly for relapsed or refactory lymphoma, following failure of standard frontline therapy. The treatment involves administration of an anti- PD-1 therapy and CD30.CAR-T cells. The treatment involves administration of at least two phases of anti-PD-1 therapy, both prior to and subsequent to, administration of CD30.CAR-T cells. The treatment may precede or include stem cell therapy such as Autologous Stem Cell Therapy (ASCT).

Description

Treatment of Lymphoma

This application claims priority from US 63/287,472 filed 8 December 2021 , the contents and elements of which are herein incorporated by reference for all purposes.

Field of the Invention

The present invention relates to methods of medical treatment and particularly, although not exclusively, to adoptive cell therapy.

Background

Classical Hodgkin lymphoma (cHL) is a neoplasm of lymphoid tissue that is histopathologically defined by the presence of malignant Reed-Sternberg (RS) cells in a background of inflammatory cells. The past few decades have seen significant progress in the management of patients with HL; it is now curable in at least 80% of patients (National Comprehensive Cancer Network [NCCN] Guideline V4, 2021). Multi-agent chemotherapy regimens used as the first and second line therapy are associated with high cure rate but are also associated with significant morbidity, including secondary malignancies, cardiac disease, pulmonary disease, and infertility (Loge et al., Ann Oncol. (1999)10, 71-77; Swerdlow et al., J Clin Oncol. (2000)18, 498-509) and non-lymphoma-related mortality (Dores et al., J Clin Oncol. (2020)38, 4149-4162; de Vries et al., J Nat Cane. Inst. (2021)113, 760-769). Furthermore, approximately 30 to 40% of patients presenting with HL will become refractory to initial therapy or will relapse. In 2021 it is estimated that there will be 8,830 new cases of HL (4,000 in females and 4,830 in males) in the United States (US) and 960 deaths (390 female and 570 males) from this disease (American Cancer Society, 2021).

HL is subdivided into classical Hodgkin lymphoma (cHL) and nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) and the immunophenotype of the malignant cells in cHL and NLPHL differs significantly. CD30 expression is characteristic of the malignant Hodgkin and RS cells that represent the pathological hallmark of cHL (Diefenbach and Leonard, Am Soc Clin Oncol Educ Book. (2012)162-166), while the lymphoid cells in NLPHL are characterized by the absence of CD30 markers (Moore et al., Hum Pathol. (2017)68, 47-53).

The standard treatment of cHL after frontline treatment failure is salvage therapy followed by consolidation with ASCT (Autologous Stem Cell Transplant). Salvage therapy options include traditional chemotherapy as well as modern regimens incorporating newer agents. Salvage chemotherapy is typically given prior to delivering high-dose chemotherapy/ASCT to maximally debulk disease. The role of ASCT in this setting is supported by two randomized studies that demonstrated improved progression- free survival (PFS) with ASCT and a trend toward better overall survival (OS; Moskowitz et al., ASCO Educational Book (2019); von Keudell and Younes, Br J Haematol. (2019)184, 105-112). Despite aggressive combination chemotherapy, between 10% and 40% of patients do not achieve a response to salvage chemotherapy. Patients achieving a metabolic complete remission (CR) on PET scans after salvage chemotherapy have a long-term relapse-free survival of approximately 75% post- ASCT. CR after salvage therapy is the critical predictor for outcome for a patient after bone marrow transplant. In contrast, patients with evidence of residual disease prior to high dose chemotherapy/ASCT have a long-term relapse-free survival that is only approximately 25%. In addition, chemotherapies used as treatment for relapse were found to be associated with short-term toxicity, long-term morbidity, and non-lymphoma-related mortality. Because results from salvage therapy directly influence long-term event-free survival post-ASCT, there is a need to develop well tolerated regimens that increase CR rates pre-ASCT.

Voorhees et al. (Blood (2019)134 (Supplement^): 3233) describe a retrospective cohort study using PD- 1 therapy following CD30.CAR-T cell therapy in 5 relapsed/refractory Hodgkin’s lymphoma patients who were heavily pre-treated with a median of 8 therapies prior to CD30.CAR-T. 4 of these patients had previously received checkpoint inhibitor therapy. All had progressive disease following the CD30.CAR-T therapy, prior to receiving anti-PD-1 therapy. The patients were not subsequently administered ASCT (i.e. after receiving anti-PD-1 therapy).

The present invention has been devised in light of the above considerations.

Summary of the Invention

In a first aspect, the present disclosure provides a method of treating a CD30-positive cancer in a subject, comprising:

(i) administering a checkpoint inhibitor therapy to the subject;

(ii) subsequently administering CD30-specific chimeric antigen receptor (CAR)-expressing T cells to the subject; and

(iii) subsequently administering a checkpoint inhibitor therapy to the subject.

In some aspects, the present disclosure provides a method of preparing a subject with a CD30-positive cancer for Autologous Stem Cell Therapy, the method comprising:

(i) administering a checkpoint inhibitor therapy to the subject;

(iii) subsequently administering a checkpoint inhibitor therapy to the subject.

The present disclosure also provides a population of CD30-specific chimeric antigen receptor (CAR) expressing T cells for use in a method of treating a CD30-positive cancer, wherein the method comprises:

(i) administering a checkpoint inhibitor therapy to the subject; (ii) subsequently administering CD30-specific chimeric antigen receptor (CAR)-expressing T cells to the subject; and

(iii) subsequently administering a checkpoint inhibitor therapy to the subject.

The present disclosure also provides the use of a population of 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:

(i) administering a checkpoint inhibitor therapy to the subject;

(iii) subsequently administering a checkpoint inhibitor therapy to the subject.

In some embodiments, the method further comprises administering stem cell therapy to the subject. The method may further comprise administering Autologous Stem Cell Therapy (ASCT) to the subject.

In some embodiments, the subject has failed a first line therapy for the CD30-positive cancer.

In some embodiments, the subject has not received any treatment for the CD30-positive cancer other than first line treatment.

In some embodiments, the checkpoint inhibitor therapy comprises an antagonist of PD-1/PD-L1 -mediated signalling. In some embodiments, the antagonist of PD-1/PD-L1 -mediated signalling is an anti-PD-1 antibody or an anti-PD-L1 antibody.

In some embodiments, the checkpoint inhibitor therapy comprises Nivolumab.

In some embodiments, the checkpoint inhibitor therapy comprises two doses of 480mg Nivolumab, administered once every four weeks.

In some embodiments, the method comprises administering 5 x 10⁷ CD30-specific CAR-expressing T cells/m² to 1 x 10⁹ CD30-specific CAR-expressing T cells/m² to the subject.

In some embodiments, the method comprises administering 1 x 10⁸ CD30-specific CAR-expressing T cells/m² to 6 x 10⁸ CD30-specific CAR-expressing T cells/m² to the subject.

In some embodiments, the method, further comprises administering lymphodepleting chemotherapy to the subject, prior to administering CD30-specific CAR-T cells to the subject.

In some embodiments, the lymphodepleting chemotherapy comprises administering fludarabine and bendamustine.

In some embodiments, the lymphodepleting chemotherapy comprises administering fludarabine at a dose of 15 to 60 mg/m² per day, for 2 to 6 consecutive days.

In some embodiments, the method comprises administering fludarabine at a dose of 30 mg/m² per day, for 3 consecutive days.

In some embodiments, the method comprises administering bendamustine at a dose of 35 to 140 mg/m² per day, for 2 to 6 consecutive days. In some embodiments, the method comprises administering bendamustine at a dose of 70 mg/m² per day, for 3 consecutive days.

In some embodiments, the method comprises:

(i) administering two doses of Nivolumab, wherein each dose comprises 480mg administered every four weeks;

(ii) administering CD30-specific CAR-expressing T cells to the subject at a dose of 2 x 10⁸ CD30- specific CAR-expressing T cells/m² to 6 x 10⁸ CD30-specific CAR-expressing T cells/m²; and

(iii) administering two doses of Nivolumab, wherein each dose comprises 480mg administered every four weeks.

In some embodiments, the method further includes:

(iv) after 8 weeks, administering Autologous Stem Cell Therapy to the subject.

In some embodiments, the method further comprises, after step (i) and before step (ii):

(a) administering fludarabine at a dose of 30 mg/m² per day and bendamustine 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, HTLV-1 -associated adult T cell leukemia/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 a relapsed or refractory CD30-positive cancer, such as relapsed or refractory Hodgkin’s lymphoma.

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 particular embodiments, the cancer is Hodgkin’s lymphoma.

In some embodiments, the subject has previously failed therapy for the CD30-positive cancer. In some embodiments, the subject has previously been treated with a first line therapy for the CD30-positive cancer. In some embodiments, the subject has previously been treated with only one therapy for the CD30-positive cancer. In some embodiments, the subject has previously been treated with no more than 1 , 2, 3, 4, 5 or 6 therapies for the CD30-positive cancer. In some embodiments, the subject has previously been treated with no more than 1 , 2 or 3 therapies for the CD30-positive cancer. In some embodiments, the method is the second, third, fourth, fifth or sixth treatment for the CD30-positive cancer, preferably the first, second or third therapy that the subject has received for the CD30-positive cancer. In some embodiments, the method is a method of salvage therapy.

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. Overview of the study design. Abbreviations: AE: adverse event; AESI: adverse event of special interest; ASCT: autologous stem cell transplant; CAR-T: CD30.CAR-T; cHL: classical Hodgkin lymphoma; CR: complete response; DOR: duration of response; ECOG: Easter Cooperative Oncology Group; EOT: End of Treatment; FDG: flurodeoxyglucose; LD: lymphodepletion; ORR: overall response rate, PD: progressive disease; PFS: progression-free survival; PR: partial response; SAE: serious adverse event; SD: stable disease

Figure 2. Overview of the study procedures. Abbreviations: AE: adverse event; AESI: adverse event of special interest; ASCT: autologous stem cell transplant; CAR-T: CD30. CAR-T; cHL: classical Hodgkin lymphoma; CR: complete response; DOR: duration of response; ECOG: Easter Cooperative Oncology Group; EOT: End of Treatment; FDG: flusrodeoxyglucose; LD: lymphodepletion; Leuka: leukapheresis; M: month; Nivo: nivolumab; PD: progressive disease; PFS: progression-free survival; PR: partial response; SAE: serious adverse event; SD: stable disease; w: week

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.

The present disclosure provides a treatment for patients with CD30+ cancers, and particularly for relapsed or refactory cHL, following failure of standard frontline therapy. The treatment may be a salvage therapy. The treatment may aim to debulk the disease. The treatment may precede, or include, Autologus Stem Cell Therapy (ASCT). The treatment involves administration of an anti-PD-1 therapy and CD30. CAR-T cells. The treatment involves administration of at least two phases of anti-PD-1 therapy, both prior to and subsequent to, administration of CD30. CAR-T cells. Without wishing to be bound by theory, it is thought that the anti-PD-1 therapy augments or enhances the efficacy of CD30. CAR-T cells.

A salvage therapy may be a second line therapy, such as a second line therapy that includes chemotherapy or immunotherapy.

CAR T expansion upregulates PD-1 expression resulting in T cell exhaustion. CAR-T cells acquire a differentiated and exhausted phenotype associated with increased expression of PD-1 (Wherry, Nat Immunol. (2011) 12, 492-499; McClanahan et al., Blood. (2015) 126, 203-211 ; Kochenderfer ef a/., J Clin Oncol. (2015) 33, 540-549). PD-1 blockade may inhibit T cell exhaustion and enhance the function of CAR-T cells. Expansion of CD30. CAR-T cells begins within a few days and peaks within the first 2 to 3 weeks post infusion. Therefore, it is important that nivolumab persists in the blood at appropriate concentrations prior to CD30. CAR-T administration. Clinical studies combining CD19 CAR T therapy with PD1/PD-L1 checkpoint inhibitors for patients with relapsed/refractory NHL prior to or post CAR-T infusion demonstrate the feasibility of the combination therapy with a manageable safety profile (Osborne et al., J Clin Oncol. (2020) 38; Jacobson et al., AACR Annual Meeting (2020) Abstract CT055; Jaeger et al., Blood. (2019) 134; Hirayama et al., Blood. (2018) 132, 1680).

Manufacturing of CAR T cells takes 6 to 8 weeks, during which patients may need bridging therapy to maintain the disease status. Nivolumab treatment cycle is planned to begin after leukapheresis, while waiting for the manufacturing of CD30.CAR-T, instead of using other bridging therapies, to enhance effect of CAR-T therapy and hence combination therapy. In terms of safety, both agents (nivolumab and CD 30 CAR T therapy) are well tolerated and have only a few overlapping toxicities. The major safety concern related to nivolumab is irAEs, while the most common drug-related AEs to LD chemotherapy and CAR-T therapy are hematologic toxicities and CRS, respectively (Younes et al., Lancet Oncol. (2016)17, 1283- 1294; Ramos et al., J Clin Oncol. (2020) 38, 3794-3804; Advani et al., Blood. (2021) (online ahead of publication)).

CD30-positive cancer

The present disclosure relates to the treatment of cancer, more particularly CD30-positive cancer. In particular, the disclosure relates to the treatment of lymphoma, and more particularly to classical Hodgkin’s lymphoma.

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 NO:1 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 NO:1 are missing. The N- terminal 18 amino acids of SEQ ID NO:1 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 NO:1 , shown in SEQ ID NO:5), a 21 amino acid transmembrane domain (positions 386 to 406 of SEQ ID NO:1 , shown in SEQ ID NO:6), and a 189 amino acid cytoplasmic domain (positions 407 to 595 of SEQ ID NO:1 , 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 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 particularly preferred aspects, the cancer is lymphoma, most particularly classical Hodgkin’s lymphoma.

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 expressed on small subsets of 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), HTLV-1- associated adult T cell leukemia/lymphoma, extra-nodal NK-T cell lymphoma, various B cell nonHodgkin’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 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) 184:105-112).

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, HTLV-1 -associated adult T cell leukemia/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, such as about 3 months or more after achieving a complete response to frontline therapy. 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 some embodiments displayed a complete response to treatment, but progressed within 3 months of completing frontline therapy.

In some aspects, the CD30-positive cancer may be relapsed or refractory with respect to treatment with chemotherapy or brentuximab vedotin. CD30-specific CARs

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

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., Cancer Res (2004) 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.

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):1116- 9, Savoldo et al. Blood (2007) 110(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). 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 for the proposed clinical trial. A total of 22 lots of CD30.CAR T Cells were manufactured using either IL-2 (11 products) or IL-7/IL-15 (11 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 (/.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.

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 Immuno (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.

In some embodiments the antigen-binding domain binds 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. In some embodiments, at least one of the target antigens for the multispecific antigen-binding domain/CAR is 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.

A target antigen other than CD30 may be any target antigen. In some embodiments, the target antigen is an antigen whose expression/activity, or whose upregulated expression/activity, is positively associated with a disease or disorder (e.g. a cancer, an infectious disease or an autoimmune disease). The target antigen is preferably expressed at the cell surface of a cell expressing the target antigen. It will be appreciated that the CAR directs effect activity of the cell expressing the CAR against cells/tissues expressing the target antigen for which the CAR comprises a specific antigen-binding domain.

In some embodiments, a target antigen may be a cancer cell antigen. A cancer cell antigen is an antigen which is expressed or over-expressed by a cancer cell. A cancer cell antigen may be any peptide/polypeptide, glycoprotein, lipoprotein, glycan, glycolipid, lipid, or fragment thereof. A cancer cell antigen’s expression may be associated with a cancer. A cancer cell antigen may be abnormally expressed by a cancer cell (e.g. the cancer cell antigen may be expressed with abnormal localisation), or may be expressed with an abnormal structure by a cancer cell. A cancer cell antigen may be capable of eliciting an immune response. In some embodiments, the antigen is expressed at the cell surface of the cancer cell (/.e. the cancer cell antigen is a cancer cell surface antigen). In some embodiments, the part of the antigen which is bound by the antigen-binding molecule described herein is displayed on the external surface of the cancer cell (/.e. is extracellular). The cancer cell antigen may be a cancer- associated antigen. In some embodiments the cancer cell antigen is an antigen whose expression is associated with the development, progression or severity of symptoms of a cancer. The cancer- associated antigen may be associated with the cause or pathology of the cancer, or may be expressed abnormally as a consequence of the cancer. In some embodiments, the cancer cell antigen is an antigen whose expression is upregulated (e.g. at the RNA and/or protein level) by cells of a cancer, e.g. as compared to the level of expression of by comparable non-cancerous cells (e.g. non-cancerous cells derived from the same tissue/cell type). In some embodiments, the cancer-associated antigen may be preferentially expressed by cancerous cells, and not expressed by comparable non-cancerous cells (e.g. non-cancerous cells derived from the same tissue/cell type). In some embodiments, the cancer- associated antigen may be the product of a mutated oncogene or mutated tumor suppressor gene. In some embodiments, the cancer-associated antigen may be the product of an overexpressed cellular protein, a cancer antigen produced by an oncogenic virus, an oncofetal antigen, or a cell surface glycolipid or glycoprotein.

Cancer cell antigens are reviewed by Zarour HM, DeLeo A, Finn OJ, et al. Categories of Tumor Antigens. In: Kufe DW, Pollock RE, Weichselbaum RR, et al., editors. Holland-Frei Cancer Medicine. 6th edition. Hamilton (ON): BC Decker; 2003. Cancer cell antigens include oncofetal antigens: CEA, Immature laminin receptor, TAG-72; oncoviral antigens such as HPV E6 and E7; overexpressed proteins: BING-4, calcium-activated chloride channel 2, cyclin-B1 , 9D7, Ep-CAM, EphA3, HER2/neu, telomerase, mesothelin, SAP-1 , survivin; cancer-testis antigens: BAGE, CAGE, GAGE, MAGE, SAGE, XAGE, CT9, CT10, NY-ESO-1 , PRAME, SSX-2; lineage restricted antigens: MARTI , Gp100, tyrosinase, TRP-1/2, MC1R, prostate specific antigen; mutated antigens: 0-catenin, BRCA1/2, CDK4, CML66, Fibronectin, MART-2, p53, Ras, TGF-pRII; post-translationally altered antigens: MUC1 , idiotypic antigens: Ig, TCR. Other cancer cell antigens include heat-shock protein 70 (HSP70), heat-shock protein 90 (HSP90), glucose-regulated protein 78 (GRP78), vimentin, nucleolin, feto-acinar pancreatic protein (FAPP), alkaline phosphatase placental-like 2 (ALPPL-2), siglec-5, stress-induced phosphoprotein 1 (STIP1), protein tyrosine kinase 7 (PTK7), and cyclophilin B. In some embodiments the cancer cell antigen is a cancer cell antigen described in Zhao and Cao, Front Immunol. 2019; 10: 2250, which is hereby incorporated by reference in its entirety. In some embodiments, a cancer cell antigen is selected from CD30, CD19, CD20, CD22, ROR1 R, CD4, CD7, CD38, BCMA, Mesothelin, EGFR, GPC3, MUC1 , HER2, GD2, CEA, EpCAM, LeY and PSCA.

In some embodiments, a cancer cell antigen is an antigen expressed by cells of a hematological malignancy. In some embodiments, a cancer cell antigen is selected from CD30, CD19, CD20, CD22, ROR1 R, CD4, CD7, CD38 and BCMA.

In some embodiments, a cancer cell antigen is an antigen expressed by cells of a solid tumor. In some embodiments, a cancer cell antigen is selected from Mesothelin, EGFR, GPC3, MUC1 , HER2, GD2, CEA, EpCAM, LeY and PSCA.

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 , CD3E, CD36, CD3y, CD3 TCRa TCR0, CD4, CD8a, CD80, 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, TL1A, 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, CD80 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 sequence. 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/l(X)e-8 YXXL/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, CD79 , 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.

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 lgG1 , 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 lgG1 .

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 (/.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 lgG1 , and an amino acid sequence which is, or which is derived from, the CH2-CH3 region (/.e. the Fc region) of human lgG1.

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., Nature Methods (2011) 8: 785-786) or Signal-BLAST (Frank and Sippl, Bioinformatics (2008) 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 (/.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, luminescent, 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 lgG1 , 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):1116-9, 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) 110(7):2620-30, Koehler et al. Cancer Res. (2007) 67(5):2265-2273, Di Stasi et al. Blood (2009) 113(25):6392-402, Hornbach et al. Gene Therapy (2010) 17:1206-1213, Chmielewski et al. Gene Therapy (2011) 18:62-72, Kofler et al. Mol. Ther. (2011) 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 (/.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 TCR ), 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)).

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 (/.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.

T cells may be activated prior to introduction of nucleic acid encoding the CAR. For example, T cells within populations of PBMCs may be non-specifically activated by stimulation in vitro with agonist anti- CD3 and agonist anti-CD28 antibodies, in the presence of IL-2.

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 vectors) 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 antagonist anti-CD3 and andti- CD28 antibodies, 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.

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 (2011), 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 ⁵¹Cr 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.

Checkpoint Inhibitor Therapy

Embodiments described here involve the administration of checkpoint inhibitor therapy. Checkpoint inhibitor therapy involves the administration of one or more agents that are capable of inhibiting signalling mediated by immune checkpoint molecules. Checkpoint inhibitors may function by blocking checkpoint proteins from binding to their partner proteins. Checkpoint proteins on the surface of T cells engage with proteins on the surface other cells, such as tumor cells, sending an “off” signal to the T cell, preventing the T cell from destroying the tumor cell. By blocking the binding of checkpoint proteins to their partner proteins, checkpoint inhibitors prevent the “off’ signal from being sent, allowing the T cells to kill the tumor cell.

Several checkpoint inhibitor therapies are known and are useful in the therapeutic methods disclosed herein. These inhibit checkpoint proteins including PD-1 , CTLA-4, LAG-3, TIM-3, TIGIT and BTLA, and checkpoint inhibitor therapies disclosed . In particular embodiments the checkpoint inhibitor is an agent capable of inhibiting signalling mediated by PD-1. The agent capable of inhibiting signalling mediated by PD-1 may be a PD-1 or PD-L1 -targeted agent. The agent capable of inhibiting signalling mediated by PD- 1 may e.g. be an antibody capable of binding to PD-1 or PD-L1 and inhibiting PD-1 -mediated signalling. In some embodiments the agent is an antagonist anti-PD-1 antibody.

Checkpoint inhibitor therapies are known in the art, and include e.g. antibodies capable of binding to immune checkpoint molecules or their ligands, and inhibiting signalling mediated by the immune checkpoint molecule. Other agents capable of inhibiting signalling mediated by an immune checkpoint molecule include agents capable of reducing gene/protein expression of the immune checkpoint molecule or a ligand for the immune checkpoint molecule (e.g. through inhibiting transcription of the gene(s) encoding the immune checkpoint molecule/ligand, inhibiting post-transcriptional processing of RNA encoding the immune checkpoint molecule/ligand, reducing stability of RNA encoding the immune checkpoint molecule/ligand, promoting degradation of RNA encoding the immune checkpoint molecule/ligand, inhibiting post-translational processing of the immune checkpoint molecule/ligand, reducing stability the immune checkpoint molecule/ligand, or promoting degradation of the immune checkpoint molecule/ligand), and small molecule inhibitors.

In some aspects, the checkpoint inhibitor therapy is an antibody that binds to the checkpoint protein. In some aspects, the checkpoint inhibitor therapy is an antibody that binds to the protein to which the checkpoint protein binds. The antibody may be an inhibitory antibody. The antibody disrupts binding between the checkpoint protein and the protein to which the checkpoint protein binds.

The PD-1 pathway is a key immune-inhibitory mediator of T-cell exhaustion. Blockade of this pathway can lead to T-cell activation, expansion, and enhanced effector functions. As such, PD-1 negatively regulates T cell responses. PD-1 has been identified as a marker of exhausted T cells in chronic disease states, and blockade of PD-1 :PD-1 L interactions has been shown to partially restore T cell function. (Sakuishi et al., JEM Vol. 207, September 27, 2010, pp2187-2194).

Several checkpoint inhibitor therapies that blockade the PD-1 pathway are known in the art and may be useful in the methods disclosed herein. Such checkpoint inhibitors may be referred to as antagonists of PD-1/PD-L1-mediated signalling. These include anti-PD-1 antibodies and anti-PD-L1 antibodies.

In some methods disclosed herein, the checkpoint inhibitor therapy is Nivolumab. In some methods, Nivolumab is administered before the CD30.CAR-T cells. In some methods, Nivolumab is administered after the CD30.CAR-T cells. In some methods, Nivolumab is administered both before and after the CD30.CAR-T cells.

Nivolumab (Opdivo™; BMS-936558) is an anti-PD-1 antibody that was approved for the treatment of melanoma in Japan in July 2014. Other anti-PD-1 and anti-PD-L1 antibodies include Pembrolizumab (Keytruda™; MSD), Ateolizumab (Tecentriq™; Genentech/Roche), Avelumab (Bavencio™; Merck KGaA and Pfizer), Durvalumab (Imfinzi™; Medimmue/AstraZeneca), Cemiplimab (Libtayo™; Regeneron) and are described in WO 2010/077634, WO 2006/121168, WO2008/156712 and WO2012/135408, the contents of which are incorporated by reference in their entirety.

Nivolumab is currently approved in the US for adult cHL patients that have relapsed or progressed after autologous HSCT and BV, or after 3 or more lines of systemic therapy that included autologous HSCT. There is also a Phase % study evaluating Brentuximab vedotin combined with nivolumab as first salvage therapy in patients with relapsed or refractory cHL. In this 3-part study, patients received staggered dosing of BV and nivolumab in the first treatment cycle, followed by same-day dosing in subsequent treatment cycles (Cycles 2 to 4) for Part 1 and 2. In Part 3, both study drugs were dosed same day for all 4 treatment cycles. In 91 evaluable patients, the overall response rate was 85%, with 67% achieving a complete response.

Nivolumab is a fully-human monoclonal antibody (immunoglobulin G4 [lgG4]) that targets PD-1 protein. In vitro, nivolumab binds to PD-1 with high affinity and inhibits the binding of PD-1 to its ligands PD-L1 and PD-L2. Nivolumab blocks the PD-1 pathway and results in a reproducible enhancement of both proliferation and interferon gamma (IFN-y) release in the mixed lymphocyte reaction. Using a CMV restimulation assay with human peripheral blood mononuclear cells (PBMC), the effect of nivolumab on antigen-specific recall response indicates that nivolumab augments IFN-y secretion from CMV-specific memory T cells in a dose-dependent manner vs. isotype-matched control. In vivo blockade of PD-1 by a murine analog of nivolumab enhances the antitumor immune response and results in tumor rejection in several immunocompetent mouse tumor models (Wolchok et al., Clin Cancer Res (2009)15, 7412-20).

Nivolumab received accelerated approval in the US for adult cHL patients that have relapsed or progressed after autologous HSCT and BV, or after 3 or more lines of systemic therapy that included autologous HSCT (Opdivo® Prescribing Information 2021). The safety of nivolumab was evaluated in 266 adult cHL patients (243 in the Checkmate-205 and 23 patients in the Checkmate-039 trials). Patients received nivolumab 3 mg/kg as an I.V. over 60 minutes every 2 weeks until disease progression, maximal clinical benefit, or unacceptable toxicity. The median age was 34 years (range: 18 to 72), 98% of patients had received autologous HSCT, none has received allogeneic HSCT, and 74% had received BV. The median number of prior systemic regimens was 4 (range: 2 to 15). Patients received a median of 23 doses (cycles) of nivolumab (range: 1 to 48), with a median duration of therapy of 11 months (range: 0 to 23 months). The most frequent serious adverse reactions reported in > 1% of patients were pneumonia, infusion-related reaction, pyrexia, colitis or diarrhea, pleural effusion, pneumonitis, and rash. The most common adverse reactions (>20%) among all patients were upper respiratory tract infection, fatigue, cough, diarrhea, pyrexia, musculoskeletal pain, rash, nausea and pruritus. The most common (>20%) treatment-emergent laboratory abnormalities included cytopenias, liver function abnormalities and increased lipase. Other common findings (>10%) included increased creatinine, electrolyte abnormalities, and increased amylase. Grade 3-4 cytopenia was reported at less than 5% (Opdivo® Prescribing Information 2021). The efficacy of nivolumab was also evaluated in 258 patients in Checkmate-205 and Checkmate-039 combined studies who had relapsed or progressive cHL after autologous HSCT. The ORR of these combined studies was 69% (95% Cl: 63, 75), with CR of 14% (95% Cl: 10, 19) and PR of 55% (95% Cl: 49, 61) (Opdivo® Prescribing Information 2021).

More recently, a Phase 1/2 study evaluated BV combined with nivolumab as first salvage therapy in patients with relapsed or refractory cHL. In this 3-part study, patients received staggered dosing of BV and nivolumab in the first treatment cycle, followed by same-day dosing in subsequent treatment cycles (Cycles 2 to 4) for Part 1 and 2. In Part 3, both study drugs were dosed same day for all 4 treatment cycles. At the end of treatment, patients could undergo ASCT per Investigator’s discretion. In 91 evaluable patients, the ORR was 85%, with 67% achieving a CR. At a median follow-up of 34.3 months, the estimated PFS rate at 3 years was 77% (95% Cl: 65% to 86%) and was even higher at 91% (95% Cl: 79% to 96%) for patients undergoing ASCT directly after study treatment. OS at 3 years was 93% (95% Cl: 85% to 97%). The most common AEs prior to ASCT were nausea (52%) and IRRs (43%), all Grade 1 or 2 in severity. A total of 16 patients (18%) had immune-related AEs (irAEs) that required systemic corticosteroid treatment (Advani et al., Blood. (2021) (online ahead of publication)).

Lymphodepleting chemotherapy

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’ required for expansion and activity of adoptively transferred lymphoid cells.

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

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

Bendamustine is an alkylating agent which causes intra-strand and inter-strand cross-links between DNA bases. It is often employed as a chemotherapeutic agent for the treatment of chronic lymphocytic leukemia, multiple myeloma and non-Hodgkin’s Lymphoma. Bendamustine is typically administered intravenously.

CD30-specific CAR-expressing T cells may be administered 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20 or more days after the administration of lymphodepleting chemotherapy. In some methods described here, CD30-specific CAR-expressing T cells are administered between 2 and 14 days after the administration of lymphodepleting chemotherapy. In some methods, CD30-specific CAR- expressing T cells may be administered about 3 days after the administration of lymphodepleting chemotherapy. Methods of treatment

The present disclosure provides methods for the treatment of cancer, particularly CD30-positive cancer and lymphoma, such as classical Hodgkin’s lymphoma.

Methods according to the invention are particularly useful for the treatment of subjects that have failed first line treatment for the disease. The terms “first line treatment”, “first line therapy”, “front line treatment” and “front line therapy” are used interchangeably herein. These terms are used to refer to the first treatment given for the disease. Such treatment is normally a standard treatment. It is often the “standard-of-care” or “proper” treatment for the disease. It is often the best available treatment for the disease. In the context of classical Hodgkin’s lymphoma, the first line treatment may include one or more of chemotherapy and radiotherapy. The first line treatment may involve administration of brentuximab vedotin (Adcentris™). The chemotherapy may be ABVD chemotherapy (doxorubicin, bleomycin, vinblastine, dacarbazine). The radiotherapy may be involved field radiation therapy. In the methods of the present disclosure, the subject has failed first line treatment. To fail first line treatment means that the subject has completed the first line treatment but has the disease. This may mean that the disease has relapsed (i.e. the first line treatment was effective, but the disease has returned after a period of time), or the disease may be refractory (i.e. the first line treatment was not effective at treating the disease, or the disease progressed despite the treatment).

The methods according to the present disclosure generally comprise administering checkpoint inhibitor therapy, subsequently administering CD30-specific CAR-expressing T cells, and subsequently administering checkpoint inhibitor therapy to a subject. Thus, the methods generally comprise a sandwich treatment in which checkpoint inhibitor therapy is administered both before and after administration of CD30-specific CAR-expressing T cells.

In some cases, the present disclosure provides a method of treating a CD30-positive cancer, such as a CD30-positive lymphoma, in a subject, the method comprising: (i) administering a checkpoint inhibitor therapy to the subject; (ii) subsequently administering CD30-specific CAR-expressing T cells to the subject; and (iii) subsequently administering a checkpoint inhibitor therapy to the subject.

The present disclosure also provides CD30-specific CAR-expressing T cells (e.g. a population of such cells) for use in a method of treating a CD30-positive cancer, such as a CD30-positive lymphoma, wherein the method comprises: (i) administering a checkpoint inhibitor therapy to the subject; (ii) subsequently administering CD30-specific CAR-expressing T cells to the subject; and (iii) subsequently administering a checkpoint inhibitor therapy to the subject. The present disclosure also provides the use of CD30-specific CAR-expressing T cells (e.g. a population of such cells) in the manufacture of a medicament for use in a method of treating a CD30-positive cancer such as a CD30-positive lymphoma, wherein the method comprises: (i) administering a checkpoint inhibitor therapy to the subject; (ii) subsequently administering CD30-specific CAR-expressing T cells to the subject; and (iii) subsequently administering a checkpoint inhibitor therapy to the subject.

The present disclosure also provides a checkpoint inhibitor agent (e.g. an anti-PD1 antibody such as Nivolumab) for use in a method of treating a CD30-positive cancer such as a CD30-positive lymphoma, wherein the method comprises: (i) administering a checkpoint inhibitor therapy (e.g. an anti-PD1 antibody such as Nivolumab) to the subject; (ii) subsequently administering CD30-specific CAR-expressing T cells to the subject; and (iii) subsequently administering a checkpoint inhibitor therapy (e.g. an anti-PD1 antibody such as Nivolumab) to the subject. The present disclosure also provides the use of a checkpoint inhibitor agent (e.g. an anti-PD1 antibody such as Nivolumab) in the manufacture of a medicament for use in a method of treating a CD30-positive cancer such as a CD30-positive lymphoma, wherein the method comprises: (i) administering a checkpoint inhibitor therapy (e.g. an anti-PD1 antibody such as Nivolumab) to the subject; (ii) subsequently administering CD30-specific CAR-expressing T cells to the subject; and (iii) subsequently administering a checkpoint inhibitor therapy (e.g. an anti-PD1 antibody such as Nivolumab) to the subject.

Administration of cells and checkpoint inhibitor therapy 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.

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 checkpoint inhibitor 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 checkpoint inhibitor 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.

In some cases, the checkpoint inhibitor therapy administered prior to the administration of CD30-specific CAR-expressing T cells is the same as the checkpoint inhibitor therapy administered after the administration of CD30-specific CAR-expressing T cells. In some cases, the checkpoint inhibitor therapy administered prior to the administration of CD30-specific CAR-expressing T cells is different to the checkpoint inhibitor therapy administered after the administration of CD30-specific CAR-expressing T cells.

In some cases, the checkpoint inhibitor therapy is an anti-PD1 antibody. In some cases, the checkpoint inhibitor therapy is Nivolumab. In some cases, an anti-PD1 antibody is administered prior to the administration of CD30-specific CAR-expressing T cells and after the administration of CD30-specific CAR-expressing T cells. In some cases, Nivolumab is administered prior to the administration of CD30- specific CAR-expressing T cells and after the administration of CD30-specific CAR-expressing T cells.

Checkpoint inhibitor therapy according to the present disclosure comprises administration of checkpoint inhibitor therapy both prior to and subsequent to the administration of CD30-specific CAR-expressing T cells. The course of checkpoint inhibitor therapy administered prior to the administration of CD30-specific CAR-expressing T cells may be the same as or different to the course of checkpoint inhibitor therapy administered subsequent to the CD30-specific CAR-expressing T cells.

The course of checkpoint inhibitor therapy administered prior to the administration of CD30-specific CAR- expressing T cells may comprise one or multiple administrations of one or more checkpoint inhibitor therapies. The course of checkpoint inhibitor therapy administered prior to the administration of CD30- specific CAR-expressing T cells may comprise administering a checkpoint inhibitor agent at a dose described herein and for a number of days described herein. Where multiple administrations of checkpoint inhibitor are administered, the checkpoint inhibitor agent doses may be administered discretely, with a period in between in which no checkpoint inhibitor agent is administered. 1 , 2, 3, 4, 5 or more doses of checkpoint inhibitor agent may be administered prior to the administration of CD30-specific CAR-expressing T cells. In some methods, 2 doses of checkpoint inhibitor agent are administered. One dose of checkpoint inhibitor agent may be administered every week, every two weeks, every three weeks, every four weeks, every five weeks, every six weeks, every seven weeks, every eight weeks, every nine weeks or every ten weeks. In some methods, one dose of checkpoint inhibitor agent may be administered every four weeks. In some methods, including where the subject is a pediatric subject, one dose of checkpoint inhibitor agent may be administered every two weeks. In some exemplary methods, two doses of checkpoint inhibitor agent are administered, with one (i.e. each) dose of checkpoint inhibitor agent administered every four weeks. In some methods described herein, lymphodepleting chemotherapy is administered after the checkpoint inhibitor agent is administered, prior to the administration of CD30-specific CAR-expressing T cells. In some methods described herein, CD30- specific CAR-expressing T cells are administered after the checkpoint inhibitor agent is administered. In some methods, the lymphodepleting chemotherapy or CD30-specific CAR-expressing T cells are administered after the dosage of checkpoint inhibitor agent is complete. In other words, where the dosage is one dose per four weeks, the lymphodepleting chemotherapy or CD30-specific CAR- expressing T cells are administered about four weeks after the checkpoint inhibitor agent is administered.

The checkpoint inhibitor therapy administered prior to the administration of CD30-specific CAR- expressing T cells, or may be administered after the patient has undergone venesection. The venesection may be to obtain cells from which to manufacture the CD30-specific CAR-expressing T cells.

The course of checkpoint inhibitor therapy administered subsequent to the administration of CD30- specific CAR-expressing T cells may comprise one or multiple administrations of one or more checkpoint inhibitor therapies. The course of checkpoint inhibitor therapy administered subsequent to the administration of CD30-specific CAR-expressing T cells may comprise administering a checkpoint inhibitor agent at a dose described herein and for a number of days described herein. Where multiple administrations of checkpoint inhibitor agent are administered, the checkpoint inhibitor agent doses may be administered discretely, with a period in between in which no checkpoint inhibitor agent is administered. 1 , 2, 3, 4, 5 or more doses of checkpoint inhibitor agent may be administered subsequent to the administration of CD30-specific CAR-expressing T cells. In some methods, 2 doses of checkpoint inhibitor agent are administered. One dose of checkpoint inhibitor agent may be administered every week, every two weeks, every three weeks, every four weeks, every five weeks, every six weeks, every seven weeks, every eight weeks, every nine weeks or every ten weeks. In some methods, one dose of checkpoint inhibitor agent may be administered every four weeks. In some exemplary methods, two doses of checkpoint inhibitor agent are administered, with one (i.e. each) dose of checkpoint inhibitor agent administered every four weeks.

In some methods described herein, the method includes a further phase of checkpoint inhibitor therapy, after the administration of checkpoint inhibitor therapy subsequent to the administration of CD30-speicifc CAR-expressing T cells. This further phase may be administered where the subject has been determined to have a partial response to the treatment or where the subject has stable or progressive disease, after the administration of checkpoint inhibitor therapy prior to the administration of CD30-specific CAR- expressing T cells, CD30-specific CAR-expressing T cells and checkpoint inhibitor therapy subsequent to the administration of CD30-specific CAR-expressing T cells. If this further phase of checkpoint inhibitor therapy is required, the checkpoint inhibitor agent may be administered at a dose described herein and for a number of days described herein. Where multiple administrations of checkpoint inhibitor agent are administered, the checkpoint inhibitor doses may be administered discretely, with a period in between in which no checkpoint inhibitor is administered. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more doses of checkpoint inhibitor agent may be administered. In some methods, 6 doses of checkpoint inhibitor agent are administered. One dose of checkpoint inhibitor agent may be administered every week, every two weeks, every three weeks, every four weeks, every five weeks, every six weeks, every seven weeks, every eight weeks, every nine weeks or every ten weeks. In some methods, one dose of checkpoint inhibitor agent may be administered every four weeks. In some exemplary methods, six doses of checkpoint inhibitor agent are administered, with one (i.e. each) dose of checkpoint inhibitor agent administered every four weeks.

By way of example, in some methods described herein, two doses of checkpoint inhibitor agent are administered, with one dose administered every four weeks. In other words, one dose of checkpoint inhibitor agent is administered, followed by a second dose of checkpoint inhibitor agent four weeks later. After the checkpoint inhibitor agent has been administered, the subject is administered a lymphodepleting chemotherapy. Where the checkpoint inhibitor agent is administered at a dosage of one dose administered every four weeks, the lymphodepleting chemotherapy is administered four weeks after the final dose of checkpoint inhibitor agent is administered. The lymphodepleting chemotherapy may comprise the administration of Fludarabine and Bendamustine. About 3 days after the administration of lymphodepleting chemotherapy, the subject is administered one dose of CD30-specific CAR-expressing T cells. About one week, or seven days, after the subject is administered the CD30-specific CAR- expressing T cells, the subject is administered with two doses of checkpoint inhibitor, with one dose administered every four weeks. After the second dosage of checkpoint inhibitor agent is complete (i.e. four weeks after the second dose is administered to the patient), End of Treatment checks are performed to determine if the subject has a complete response, partial response, stable disease or progressive disease. The subject may then be selected for treatment with Autologous Stem Cell Therapy (ASCT), or for a further phase of checkpoint inhibitor administration.

In some embodiments, the checkpoint inhibitor agent is Nivolumab and is administered at a dose 400mg- 550mg, 450-500mg, 470mg-490mg or about 480mg. The dose may be 1 mg-5mg/kg, 2mg-4mg/kg or about 3 mg/kg. The dose may be a fixed dose (i.e. the same dose is administered to each adult subject, independent of the size or other characteristics of the subject). The dose may be vary depending on the size of the patient. In some cases, the dose may be a fixed dose for an adult subject. The dose may vary depending on the size of the subject for a pediatric subject.

The dosage may be 1 , 2, 3, 4 or 5 doses of 400mg-550mg every 2 weeks, 450-500mg every 2 weeks, 470mg-490mg every 2 weeks, about 480mg every 2 weeks, 400mg-550mg every 3 weeks, 450-500mg every 3 weeks, 470mg-490mg every 3 weeks, about 480mg every 3 weeks, 400mg-550mg every 4 weeks, 450-500mg every 4 weeks, 470mg-490mg every 4 weeks, about 480mg every 4 weeks, 400mg- 550mg every 5 weeks, 450-500mg every 5 weeks, 470mg-490mg every 5 weeks, about 480mg every 5 weeks, 400mg-550mg every 6 weeks, 450-500mg every 6 weeks, 470mg-490mg every 6 weeks or about 480mg every 6 weeks. In particular methods described herein, the dosage is 2 doses of about 480mg every 4 weeks.

The dosage may be 1 , 2, 3, 4 or 5 doses of 1 mg-5mg/kg every 4 weeks, 2mg-4mg/kg every 4 weeks, about 3 mg/kg every 4 weeks, 1 mg-5mg/kg every 3 weeks, 2mg-4mg/kg every 3 weeks, about 3 mg/kg every 3 weeks, 1 mg-5mg/kg every 2 weeks, 2mg-4mg/kg every 2 weeks, about 3 mg/kg every 2 weeks, 1 mg-5mg/kg every 1 week, 2mg-4mg/kg every 1 week, or about 3 mg/kg every 1 week. In particular methods described herein, the dosage is 2 doses of about 3mg/kg every 2 weeks.

Particular exemplary embodiments of methods of treatment in accordance with the present disclosure are described below.

In some embodiments, the method comprises:

(ii) administering CD30-specific CAR-expressing T cells to the subject at a dose of 1 x 10⁸ cells/m²; and

Optionally, the method further includes:

(iv) after 8 weeks, administering Autologous Stem Cell Therapy to the subject.

In some embodiments, the method comprises:

(ii) administering CD30-specific CAR-expressing T cells to the subject at a dose of 1 x 10⁸ cells/m²;

(iii) administering two doses of Nivolumab, wherein each dose comprises 480mg administered every four weeks; and

(iv) administering six doses of Nivolumab, wherein each dose comprises 480mg administered every four weeks.

(v) optionally, after 8 weeks, administering Autologous Stem Cell Therapy to the subject.

In some embodiments, the method comprises:

(ii) administering fludarabine at a dose of 30 mg/m²/day and bendamustine at a dose of 70 mg/m²/day to a subject for 3 consecutive days

(iii) administering CD30-specific CAR-expressing T cells to the subject at a dose of 1 x 10⁸ cells/m²; and

(iv) administering two doses of Nivolumab, wherein each dose comprises 480mg administered every four weeks; and

The methods may include a step of isolating or obtaining a population of immune cells comprising T cells (e.g. PBMCs) from the subject. The methods may include a step of modifying a T cell to express or comprise a CD30-specific CAR, or to express or comprise nucleic acid encoding a CD30-specific CAR.

CD30-specific CAR-expressing T cells

Aspects of the present disclosure also comprise administering CD30-specific CAR-expressing T cells to a subject. The methods therefore involve adoptive cell transfer.

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;

(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 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 (/.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 (/.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 vivo 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 vivo 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 leukapheresis or venesection, 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 some embodiments, the blood sample is the source of the cells from which CD30-specific CAR-expressing T cells are prepared. In some embodiments, the blood sample is the source of the cells for autologous stem cell transplant. In some embodiments, the blood sample is the source of cells for both the preparation of CD30-specific CAR-expressing T cells and for autologous stem cell transplant.

In some methods according to 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. In some embodiments, CD30-specific CAR-expressing T cells are administered to a subject within around 3 days of completion of a course of lymphodepleting chemotherapy described herein.

In some embodiments, CD30-specific CAR-expressing T cells are administered at a dose of 1 x 10⁷ cells/m² to 1 x 10⁹ cells/m², e.g. one of 5 x 10⁷ cells/m² to 1 x 10⁹ cells cells/m², 1 x 10⁸ cells cells/m² to 9 x 10⁸ cells/m², 2 x 10⁸ cells/m² to 8 x 10⁸ cells/m², or 2 x 10⁸ cells/m² to 8 x 10⁸ cells/m². In some embodiments, CD30-specific CAR-expressing T cells are administered at a dose of 1 x 10⁸ cells/m² to 6 x 10⁸ cells/m².

In some embodiments, CD30-specific CAR-expressing T cells are administered at a dose of 2 x 10⁸ cells/m². In some embodiments, CD30-specific CAR-expressing T cells are administered at a dose of 4 x 10⁸ cells/m². In some embodiments, CD30-specific CAR-expressing T cells are administered at a dose of 6 x 10⁸ cells/m².

In some embodiments, CD30-specific CAR-expressing T cells are administered at a dose greater than 1 x 10⁸ cells/m², e.g. a dose greater than 2 x 10⁸ cells/m², 3 x 10⁸ cells/m², 4 x 10⁸ cells/m², 5 x 10⁸ cells/m², 6 x 10⁸ cells/m², 7 x 10⁸ cells/m², 8 x 10⁸ cells/m². Such embodiments are contemplated in particular where the cancer to be treated is non-Hodgkin’s Lymphoma. In some embodiments, CD30-specific CAR- expressing T cells are administered at a dose of 2 x 10⁸ cells/m² to 8 x 10⁸ cells/m². In some embodiments, CD30-specific CAR-expressing T cells are administered at a dose of 3 x 10⁸ cells/m² to 8 x 10⁸ cells/m². In some embodiments, CD30-specific CAR-expressing T cells are administered at a dose of 4 x 10⁸ cells/m² to 8 x 10⁸ cells/m². In some embodiments, CD30-specific CAR-expressing T cells are administered at a dose of 5 x 10⁸ cells/m² to 8 x 10⁸ cells/m². In some embodiments, CD30-specific CAR- expressing T cells are administered at a dose of 6 x 10⁸ cells/m² to 8 x 10⁸ cells/m².

In some embodiments, CD30-specific CAR-expressing T cells are administered at a dose of 1 x 10⁶ to 1 x 10⁷ cells per kg body weight, e.g. one of 1 .5 x 10⁶ to 9 x 10⁶ cells per kg body weight, 2.0 x 10⁶ to 8 x 10⁶ cells per kg body weight, 2.0 x 10⁶ to 6 x 10⁶ cells per kg body weight or 2.0 x 10⁶ to 5 x 10⁶ cells per kg body weight. Administration of doses calculated in this manner is contemplated in particular where the subject to be treated weighs 50 kg or less.

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.

Multiple (e.g. 2, 3, 4 or more) 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, 11 , 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.

Lymphodepleting chemotherapy

Certain methods described herein involve a lymphodepleting chemotherapy. The lymphodepleting chemotherapy may comprise administering fludarabine and bendamustine.

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 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, 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 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 bendamustine may be administered simultaneously or sequentially. Simultaneous administration refers to administration together, for example as a pharmaceutical composition containing both agents (/.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 bendamustine 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 bendamustine at a dose of 70 mg/m² per day for 3 consecutive days, the fludarabine and bendamustine 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 bendamustine 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.

Autologous Stem Cell Transplant (ASCT)

In some embodiments of the methods disclosed herein, the subject is treated with autologous stem cell transplant. In some embodiments, the subject is treated with an allogeneic stem cell transplant. In some cases, the methods disclosed herein are suitable for preparing a subject for a stem cell transplant, such as an autologous or allogeneic stem cell transplant.

Autologous stem cell transplant involves collecting blood forming cells from the subject to be treated, treating the patient, and administering the collected cells to the subject. ASCT may involve the isolation of peripheral blood stem cells (PBSCs) from blood. The method may involve obtaining a blood sample from the subject prior to treatment of the subject with a method disclosed herein. The method may involve obtaining a blood sample from the subject prior to treatment of the subject with chemotherapy, such as lymphodepleting chemotherapy. The method may involve obtaining a blood sample from the subject prior to treatment of the subject with checkpoint inhibitor therapy.

Allogeneic stem cell transplant involves transferring stem cells from a healthy donor to the subject to be treated.

Methods disclosed herein may involve treating the subject with stem cell transplant after administering checkpoint inhibitor therapy, CD30-specific CAR-expressing T cells and checkpoint inhibitor therapy to the subject. Methods disclosed herein may involve the administration of peripheral blood stem cells to the subject after administering checkpoint inhibitor therapy, CD30-specific CAR-expressing T cells and checkpoint inhibitor therapy to the subject. The method may involve the administration of peripheral blood stem cells to the subject after the further phase of checkpoint inhibitor therapy. In some embodiments, the method involves treating the subject with an autologous stem cell transplant after administering checkpoint inhibitor therapy, CD30-specific CAR-expressing T cells and checkpoint inhibitor therapy to the subject.

Subjects

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 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. In particular aspects, the subject has lymphoma, such as classical Hodgkin’s lymphoma.

The subject may have been diagnosed with a CD30-positive cancer through testing of an archived tumor tissue sample. The subject may have been diagnosed with a CD30-positive cancer through testing of a fresh tumor sample. CD30 may have been assessed and confirmed by a local pathologist in a Clinical Laboratory Improvement Amendments (CLIA)-certified or College of American Pathologist (CAP)-certified pathology laboratory.

The subject may have at least one lesion. 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 lesion may be flurodeoxyglucose postrion emission tomography (FDG-PET) avid and be measurable bidimensionally to be at least 15mm in the longest axis for nodal leions or at least 10mm for extranodal lesions (e.g. hepatic nodules) as documented by radiographic technique (i.e. PET-CT scan)

The subject has been treated for the cancer. The subject has been treated with a first line therapy. The subject may have failed first line therapy. The subject may have been treated with chemotherapy and radiotherapy. The subject may have been treated with brentuximab vedotin (Adcentris™). The chemotherapy may be ABVD chemotherapy (doxorubicin, bleomycin, vinblastine, dacarbazine). The radiotherapy may be involved field radiation therapy.

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 have responded to a treatment for the cancer, but the cancer has subsequently re-emerged/progressed after 3 months or more after achieving a complete response to first line therapy. The subject may have achieved a complete response to frontline therapy, but progressed 3 months or more after completing the frontline therapy.

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 have displayed a complete response to the frontline therapy, but the cancer has progressed within three months of completing the frontline therapy. The subject may never haver achieved a complete response to the frontline therapy.

The subject may be a subject that has previously received treatment for the cancer. The subject may have received 1 , 2, 3, 4, 5 or 6 previous therapies for the cancer. The subject may have received no more than 1 , 2, 3, 4, 5 or 6 previous therapies for the cancer. The subject may have received only first line treatment for the cancer prior to undergoing the methods disclosed herein.

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 CfD30-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 forthe 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.

The subject may be autogeneic/autologous with respect to the source of the cells administered during the ASCT treatment.

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 may result in the debulking of the disease. Debulking means to remove of as much of a tumor as possible, or to reduce the size of a tumor as much as possible. Debulking may increase the chance that a therapy will kill all the tumor cells. Debulking may increase the chance of Autologous Stem Cell Transplant resulting in a complete or partial response. The method may result in the reduction if size of at least one lesion of the subject. The size of a node or nodal mass may reduce, as compared to size of that node or nodal mass prior to the treatment. In some cases, the size of the node or nodal mass decreases to <2.5cm, <2.4cm, <2.3cm, <2.2cm, <2.1cm, <2.0cm, <1.9cm, <1.8cm, <1.7cm, <1.6cm, <1.5cm, <1.4cm, <1.3cm, <1.2cm, <1.1cm, <1.0cm, <0.9cm, <0.8cm, <0.7cm, <0.6cm, <0.5cm, <0.4cm, <0.3cm, <0.2cm or <0.1cm LDi (longest transverse diameter of a lesion). Preferably, the size of the node or nodal mass decreases to <1.5cm, <1.4cm, <1.3cm, <1.2cm, <1.1 cm, <1.0cm, <0.9cm, <0.8cm, <0.7cm, <0.6cm, <0.5cm, <0.4cm, <0.3cm, <0.2cm or <0.1 cm LDi. Preferably, the size of the node or nodal mass decreases to <1.5cm. Methods for measuring the size of nodes or nodal masses are known in the art and include PET-CT scans. In some cases, the treatment results in at least a 50% decrease in SPD (sum of the product of the perpendicular diameters for multiple lesions) of up to 6 target measurable nodes and extranodal sites. The treatment may result in a 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% or 5% decrease in SPD of up to 6 target measurable nodes and extranodal sites.

Treatment of a subject in accordance with the methods of the present disclosure may result in a Complete metabolic response in the lymph nodes or extralymphatic sites. A complete metabolic response is a return of FDG uptake in previously documented lesions to a level equivalent to, or less than, residual radioactivity in normal tissues within the organ in question. In some cases, complete metabolic response is indicated by no uptake of FDG above background, uptake of FDG above background but less than uptake of FDG by the mediastinium, or uptake above the mediastinium but less than the uptake of FDG by the liver. In some cases, the methods of the present disclosure result in a partial metabolic response in the lymph nodes or extralymphatic sites. A partial metabolic response is a reduction in FDG uptake as compared to the level of FDG uptake prior to the treatment. A partial metabolic response may be indicated by an uptake of FDG that is moderately greater than the level of FDG uptake by the liver, or an uptake of FDG that is markedly higher than the level of FDG uptake by the liver and/or new lesions, but which is lower than the FDG uptake prior to the treatment.

Treatment of a subject in accordance with the methods of the present disclosure may result in the absence in the presence of FDG-avid (fluorodeoxyglucose-avid) disease in bone marrow. In some cases, the FDG-avid disease was present in the marrow of the subject prior to the treatment, but is absent in the subject after the treatment. In some cases, the amount of FDG-avid disease in the marrow of the subject is reduced by the treatment. In some cases, the amount of FDG-avid disease in the marrow of the subject is higher than uptake in normal marrow (i.e. marrow of the patient that is not diseased, or marrow from a comparable subject that does not have the disease), but is lower than the amount of FDG-avid disease in the marrow of the subject prior to the treatment. Preferably, the treatment results in the absence of FDG-avid disease in the marrow.

Methods for determining the presence, absence or level of FDG-avid disease in marrow are known in the art and include fluordeoxyglucose positron emission tomography (FDG-PET).

The methods disclosed herein may also have one or more of the following effects: reduced number of CD30-positive cancer cells in the subject, inhibition (e.g. prevents or slows) of growth of CD30-positive cancer cells in the subject, inhibition (e.g. prevents or slows) of growth of a CD30-positive tumor/lesion in the subject, inhibition (e.g. prevents or slows) of the development/progression of a CD30-positive cancer (e.g. to a later stage, or metastasis), reduction of the severity of symptoms of a CD30-positive cancer in the subject, increase in survival of the subject (e.g. progression free survival or overall survival), reduced correlate of the number or activity of CD30-positive cancer cells in the subject, and/or reduced 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, 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 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, 11 , 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.

Pairwise and multiple sequence alignment for the purposes of determining percent identity between two 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 (Sbding, 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 parameters, e.g. for gap penalty and extension penalty, are preferably used.

Sequences

***

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%.

Where a nucleic acid sequence is disclosed herein, the reverse complement thereof is also expressly contemplated.

Methods described herein may be performed in vitro or in vivo. In some embodiments, methods described herein are performed in vitro. The term “in vitro" is intended to encompass experiments with cells in culture whereas the term “in vivo" is intended to encompass experiments with intact multi-cellular organisms. Examples

EXAMPLE 1: Phase 2 Study Evaluating the Safety and Efficacy of Autologous CD30.CAR-T in Combination with Programmed Cell Death Protein-1 Checkpoint Inhibitor (Nivolumab) in Relapsed or Refractory Classical Hodgkin Lymphoma Patients after Failure of Frontline Therapy

1.1: Study Rationale

The standard treatment of cHL after frontline treatment failure is salvage therapy followed by consolidation with ASCT (Moskowitz et al, 2019). Salvage chemotherapy is typically given prior to delivering high-dose chemotherapy/ASCT to maximally debulk disease. Despite aggressive combination chemotherapy, between 10% and 40% of patients do not achieve a response to salvage chemotherapy (von Keudell and Younes, Br J Haematol. (2019) 184, 105-112). Patients achieving a metabolic CR on PET scans after salvage chemotherapy have a long-term relapse-free survival of approximately 75% post-ASCT. In contrast, patients with evidence of residual disease prior to high dose chemotherapy/ASCT have a long-term relapse-free survival that is only approximately 25% (Moskowitz et al., Br J Haematol. (2010) 148, 890-897.). In addition, chemotherapies used as treatment for relapse were found to be associated with short-term toxicity, long-term morbidity, and non-lymphoma-related mortality (Dores et al., J Clin Oncol. (2020) 38, 4149-4162; de Vries et al., J Nat Cane Inst. (2021) 113, 760 769). Because results from salvage therapy directly influence long-term event-free survival post-ASCT, it is critical to develop well tolerated regimens that increase CR rates pre-ASCT (Moskowitz et al., Blood (2012) 119, 1665-1670).

Newer approaches including CD30-targeted therapy (Younes et al., J Clin Oncol (2012) 20, 2183-2189; Gopal et al., Blood (2015) 125, 1236-1243; Moskowitz et al., Lancet (2015) 385, 1853-1862; O’Connor et al., Lancet Oncol (2018) 19, 257-266; Ramos et al., J Clin Oncol (2020) 38, 3794-3804) and PD-1 checkpoint inhibitors (Younes et al., Lancet Oncol (2016) 17, 1283-1294; Chen et al., J Clin Oncol (2017) 35, 2125 2132; Armand et al., J Clin Oncol (2018) 36, 1428-1439; Kuruvilla et al., J Clin Oncol (2020) 38, 8005) have been shown to be effective and safe for cHL therapy. Consequently, management of relapsed and refractory cHL has changed substantially since the approval of BV (an anti-CD30 ADC) along with the PD-1 checkpoint inhibitors nivolumab and pembrolizumab (Moskowitz et al., ASCO Educational Book (2019)). Furthermore, combination therapy of PD-1 checkpoint inhibitors and CD30-directed therapy as second line treatment of cHL is a promising novel regimen, demonstrating high clinical efficacy and acceptable safety (Advani et al., Blood. (2021) (online ahead of publication)). In a Phase 1/2 study (NCT02572167), BV combined with nivolumab as first salvage therapy in 91 patients with relapsed or refractory cHL showed an ORR of 85%, with 67% of the patients achieving a CR. The most common AEs prior to ASCT were nausea (52%) and IRR (43%), which were Grade 1 or 2 in severity. There were 16 (18%) patients who experienced irAEs that required systemic corticosteroid treatment (Advani et al., Blood. (2021) (online ahead of publication)).

CD30-directed CAR-T cell therapy has already demonstrated clinical efficacy with a favorable safety profile in 2 parallel Phase 1/2 studies (NCT02690545 and NCT02917083; Ramos et al., J Clin Oncol (2020) 38, 3794-3804). Heavily pre-treated patients with relapsed or refractory cHL who received fludarabine-based LD chemotherapy followed by CD30. CAR-T infusion had a high rate of durable responses and an excellent safety profile. The ORR in 32 evaluable patients with active disease was 72%, including 19 patients (59%) who achieved CR (Ramos et al., J Clin Oncol (2020) 38, 3794-3804).

The potential synergism between PD-1 checkpoint inhibitors and CD30.CAR-T cell therapy have been shown in pre-clinical studies where anti-PD-1 or PD-L1 antibodies can boost CAR-T cell therapy and promote increased tumor cell death in vivo (Cherkassky et al., J. Clin Investig. (2016) 126, 3130-3144; Moon et al., Clin Cancer Res. (2014) 20, 4262-4273; Gargett et al., Mol Ther. (2016) 24, 1135-1149.). These preclinical studies suggest that stimulation of CAR may escalate the expression of PD-1 inhibitory signalling, and interference of PD-1 pathway may restore the effector function of CAR-T cells, indicating that PD-1 blockade would be an effective strategy in improving the potency of CAR-T cell therapy.

Furthermore, a retrospective cohort study evaluating safety of PD-1 therapy following CD30. CAR-T cell therapy in 5 relapsed/refractory HL patients who were heavily pretreated with a median of 8 therapies prior to CD30. CAR-T demonstrated clinical efficacy of PD-1 checkpoint inhibitor after CD30. CAR-T even in those patients who had progressed after prior treatment with PD-1 checkpoint inhibitor. All patients in this cohort achieved an objective response with 4 of the 5 patients achieving a CR and 3 patients achieving an improved response to the PD-1 checkpoint inhibitor compared to when they initially received the same drug prior to CD30. CAR-T therapy (Voorhees et al., Blood (2019) 134, 3233). These patients were at a very advanced stage of disease (extensive prior therapeutic lines).

This study will investigate a potential synergistic effect of CD30. CAR-T therapy in combination with a PD- 1 checkpoint inhibitor, nivolumab, as salvage therapy. In contrast to the Voorhees et al., 2019 study, the treatment will be investigated at an earlier stage of disease. As a salvage therapy, the aim of the treatment is to debulk disease, prior to Autologous Stem Cell Transplant (ASCT) therapy. An overview of the study design is set out in Figure 1 .

1.2 Objectives and End points

Primary Objectives

To assess CR rate of autologous CD30. CAR-T in combination with nivolumab, at EOT (after 4 treatment cycles of nivolumab and a single infusion of CD30. CAR-T) using the Lugano Classification Revised Staging System for malignant lymphoma (Cheson et al., J Clin Oncol. (2014) 32, 3059-3068).

Secondary Objectives

The following will also be assessed:

• the safety profile of autologous CD30. CAR-T in combination with the PD-1 checkpoint inhibitor, nivolumab

• Objective Response Rate (ORR), Duration of response (DOR), and Progression Free Survival (PFS) after ASCT (ASCT patients)

ORR, DOR, and PFS for patients not treated with ASCT (non-ASCT patients) Exploratory Objectives

These include:

• Overall Survival (OS) for all patients

• Expansion and persistence of autologous CD30.CAR-T in blood

• Immunogenicity against anti-CD30 scFv in blood following CD30.CAR-T infusion

• Cytokine profiling in blood

• Immunological parameters in blood

• Circulating tumor DNA (ctDNA) in blood

• Tumor markers in blood and tumor tissue

1.3 Study Population

The study population includes male or female patients who are 12 to 75 years of age (inclusive) with relapsed or refractory cHL who have failed a standard frontline chemotherapy. Relapsed disease is defined as achieving a complete response (CR) to frontline therapy, but then progressing 3 months or more after completing frontline therapy. Refractory disease is defined as never achieving a CR to frontline therapy or achieving a CR but then progressing within 3 months of completing frontline therapy.

Patients must have at least one lesion that is fluorodeoxyglucose postiron emission tomography (FDG- PET) avid and that measures bidimensionally to be at least 15mm in the longest axis for nodal lesions or at least 10mm for extranodal lesions (e.g. hepatic nodules) as documented by radiographic technique (i.e. PET-CT scan).

Patients must also have documented CD30-positivity, either from previous documentation, CD30 testing of an archived tumor tissue sample (if previous documentation is not available), or CD30 testing of a fresh tumor tissue sample (if an archival sample is not available). CD30 expression on fresh or archived tumor tissue will be assessed and confirmed by a local pathologist in a Clinical Laboratory Improvement Amendments (CLIA)-certified or College of American Pathologis (CAP)-certified pathology laboratory.

1.4 Study Design

This is a Phase 2, single arm study to evaluate the safety and efficacy of the combination therapy, CD30.CAR-T and the PD-1 checkpoint inhibitor nivolumab. Approximately 43 adult and pediatric patients with relapsed or refractory cHL following failure of standard frontline therapy will be enrolled in the study.

1.5 Study Treatment

An overview of the study procedures is provided in Figure 2. Screening assessments must be completed and the patient confirmed eligible within 28 days prior to leukapheresis. Leukapheresis will then be performed for the production of CD30.CAR-T.

Four (4) nivolumab treatment cycles (480 mg) will be administered Q4W; 2 nivolumab treatment cycles prior to LD chemotherapy/CD30.CAR-T administration, and 2 nivolumab treatment cycles post- CD30.CAR-T infusion. Pre-nivolumab assessments will be performed within 3 days prior to each cycle of nivolumab administration. Nivolumab Cycle 1 will start after ? days post leukapheresis, followed by nivolumab Cycle 2, which will be administered 4 weeks after nivolumab Cycle 1 . In case of toxicity, a delay in the next treatment cycle will be adjusted according to the nivolumab prescribing information (Opvido® Prescribing Information 2021).

All patients will have disease status and safety assessments repeated, as pre-LD assessments prior to starting LD chemotherapy. Pre-LD assessments will begin between 3 to 4 weeks post nivolumab Cycle 2 administration unless there is toxicity due to nivolumab. Imaging scans for disease assessment will be performed up to 7 days prior to LD chemotherapy; safety assessments should be performed within 3 days prior to LD chemotherapy. Upon meeting the treatment criteria for LD chemotherapy, patients will undergo LD chemotherapy for 3 consecutive days with bendamustine 70 mg/m2/day as an IV infusion over 10 or 30 minutes and fludarabine 30 mg/m2/day IV over 30 minutes. Bendamustine is recommended to be infused first, followed by fludarabine.

Prior to CD30.CAR-T infusion, safety assessments will be performed. Blood samples for hematology, biochemistry, and coagulation panels will be tested within 2 days prior to CD30.CAR-T administration. CD30.CAR-T infusion will be administered on the third day after completion of LD chemotherapy (A window of 3 to 14 days is allowed in the event of scheduling difficulties including weekends, holidays, need for toxicity recovery, or other unforeseen events, if indicated and after discussion with the Sponsor or designee).

Patients will receive CD30.CAR-T as an IV infusion over 30 minutes at a dose of 2 x 10⁸ CD30.CAR expressing T cells/m2 of body surface area (BSA). The allowable dose range at the time of release is 2.0 to 2.7 x 10⁸ CD30.CAR-T cells/m² of BSA. If the dose exceeds 2.7 x 10⁸ CD30.CAR-T cells/m² of BSA at the time of product release, the dose will be adjusted to 2.4 x 10⁸ CD30.CAR-T cells/m² of BSA prior to infusion. If the dose is below 2.0 x 10⁸ cells/m² but above 1 .0 x 10⁸ cells/m² at the time of product release, the batch will be released for infusion. For patients with a BSA > 2.4 m², the dose to be administered will be based on a maximum BSA of 2.4 m².

Following LD and CD30.CAR-T administration, nivolumab Cycle 3 and Cycle 4 will be administered (Q4W). Nivolumab Cycle 3 should be administered 1 week following CD30.CAR-T infusion, which will be 5 to 6 weeks post- nivolumab Cycle 2. Nivolumab Cycle 4 should be administered 4 weeks after nivolumab Cycle 3. In case of toxicity, a delay in the next treatment cycle will be adjusted according to the nivolumab prescribing information (Opvido® Prescribing Information 2021).

LD chemotherapy and CD30.CAR-T infusion can be administered as an outpatient or patients may be hospitalized for the administration of LD chemotherapy and/or CD30.CAR-T infusion per institutional guidance. Post-CD30.CAR-T infusion, patients are required to stay in the treating facility for an additional 4 hours. Per institutional guidance, patients may remain hospitalized topost-CD30.CAR-T infusion for toxicity monitoring. The extended hospitalization will not be considered a SAE.

Patients will be monitored daily for the first 14 days following CD30.CAR-T infusion, followed by weekly follow-up until post-CD30.CAR-T week 6, and then at EOT, at the end of post CD30.CAR-T week 8. At EOT (end of post-CAR-T Week 8), patients will undergo response assessments. If pseudoprogression is suspected, a confirmatory PET-CT scan is required after 4 to 6 weeks, with tumor biopsy for further confirmation of disease progression vs. pseudoprogression.

Based on Investigator’s decision, patients who have a response assessment of either CR, PR, or SD at the EOT visit will undergo ASCT or continue to receive treatment with nivolumab. After EOT, ASCT will be scheduled for eligible patients while non-ASCT patients (based on Investigator’s decision) will continue to receive nivolumab 480 mg for 6 additional treatment cycles (Cycle 5 up to Cycle 10) unless patients experience either PD or unacceptable toxicity is reported, whichever occurs earlier. Non-ASCT patients who experience CR and PR following continued treatment with nivolumab can also be considered for ASCT, after discussion between Sponsor and Investigator.

All patients will be evaluated for safety and efficacy after CD30.CAR-T infusion throughout the Treatment Phase (until EOT) and Post-treatment Follow-up (FU; until the EOS) phases according to the schedules provided in Section 7.3 and Section 7.4. ASCT patients will be followed-up every 3 months (Q3M) from post-EOT M3, followed by post-EOT M6, M9, M12, M15, M18, M21 , M24 and M27)until post-EOT M30 (EOS. Non-ASCT patients will continue to receive nivolumab 480 mg Q4Wfrom EOT for up to 6 treatment cycles (Cycle 5 up to Cycle 10) unless patients experience either PD or unacceptable toxicity is reported, whichever occurs earlier. Pre-nivolumab assessments will be performed within 3 days prior to nivolumab infusion. Response assessments for non-ASCT patients will be performed after 3 treatment cycles (every 3 months; Q3M) beginning with nivolumab Cycle 8 and then post- nivolumab Month 1 (M1). Patients will continue to be followed-up at Q3M from the last cycle of nivolumab (up to Cycle 10, unless patients experience either PD or unacceptable toxicity is reported, whichever occurs earlier) at post- nivolumab M3, M9, M12, M15, M18, M21 , until post-nivolumab M24 (EOS, i.e., end of Y3).

Safety monitoring, including AE and SAE, and adverse events of special interest (AESI) collection will begin from the time of signing the informed consent form (ICF) through to the Long-term Follow-up (LTFU) phase. At the LTFU, which will include survival FU, safety monitoring will occur Q6M from EOS until Y5, then annually thereafter from Y5 to Y15 (refer to Section 7.5). A safety monitoring committee (SMC) will be established to have proper safety oversight in this study.

Imaging scans will be reviewed for response assessment from Screening by the Investigator, and by an independent radiologist (as appropriate). Response assessments will be performed according to the Lugano Classification Revised Staging System for malignant lymphoma (Cheson et al., J Clin Oncol (2014)32, 3059-3068). Response assessments by positron emission tomography-computed tomography (PET-CT) scans will be performed at Screening (as baseline), prior to LD chemotherapy and at the EOT visits. After EOT, response assessments for ASCT patients will be performed Q3M until post-EOT M24, then Q6M until EOS, if clinically indicated. For non-ASCT patients who have continued nivolumab treatment, response assessment will be performed Q3M from EOT until EOS. Comparison will be made to imaging scans obtained at the Screening/Baseline assessment. If pseudoprogression is suspected, a confirmatory PET-CT scan is required after 4 to 6 weeks, with tumor biopsy for further confirmation of true disease progression vs. pseudoprogression.

Patients will undergo exploratory biomarker assessments to characterize the pharmacokinetic properties (expansion and persistence) as well as the immunogenicity and immunological properties of autologous CD30.CAR-T. Details on prioritization of blood samples collected for analysis will be included in the Laboratory Manual. Blood and tissue biopsy samples will be collected, if clinically feasible as assessed by the Investigator, at various time points before and during study treatment (refer to Appendix 1). These data will be used to analyze further the mechanisms that govern therapeutic outcome as well as to identify potential biomarkers that correlate with therapeutic efficacy and/or failure.

Following EOT, patients with CR, PR, or SD will undergo either ASCT (standard treatment) or continued treatment with nivolumab based on Investigator’s decision. For ASCT patients, ASCT will be scheduled. For non-ASCT patients, nivolumab will continue to be administered Q4W, starting after EOT for up to 6 treatment cycles (Cycle 5 up to Cycle 10) unless patients experience either PD or unacceptable toxicity, whichever occurs earlier. Patients who experience CR and PR following continued treatment with nivolumab can be considered for ASCT, based on discussion between Sponsor and Investigator.

References

A number of publications are cited above in order to more fully describe and disclose the invention and the state of the art to which the invention pertains. The entirety of each of these references is incorporated herein.

For standard molecular biology techniques, see Sambrook, J., Russel, D.W. Molecular Cloning, A

Laboratory Manual. 3 ed. 2001 , Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press

Claims

57

Claims:

1 . A method of treating a CD30-positive cancer in a subject, comprising:

(i) administering a checkpoint inhibitor therapy to the subject;

(iii) subsequently administering a checkpoint inhibitor therapy to the subject.

2. A population of CD30-specific chimeric antigen receptor (CAR)-expressing T cells for use in a method of treating a CD30-positive cancer, wherein the method comprises:

(i) administering a checkpoint inhibitor therapy to the subject;

(iii) subsequently administering a checkpoint inhibitor therapy to the subject.

3. Use of a population of 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:

(i) administering a checkpoint inhibitor therapy to the subject;

(iv) subsequently administering a checkpoint inhibitor therapy to the subject.

4. The method according to claim 1 , the population for use according to claim 2, or the use according to claim 3, wherein the method further comprises administering stem cell therapy to the subject, such as Autologous Stem Cell Therapy.

5. The method according to claim 1 , the population for use according to claim 2, or the use according to claim 3, wherein the subject has failed a first line therapy for the CD30-positive cancer.

6. The method, the population for use or the use according to claim 5, wherein the subject has not received any treatment for the CD30-positive cancer other than first line treatment.

7. The method, the population for use or the use according to any one of the preceding claims, wherein the checkpoint inhibitor therapy comprises an antagonist of PD-1/PD-L1 -mediated signalling, optionally wherein the antagonist of PD-1/PD-L1-mediated signalling is an anti-PD-1 antibody or an anti-PD-L1 antibody.

8. The method, the population for use or the use according to any one of the preceding claims, wherein the checkpoint inhibitor therapy comprises Nivolumab. 58

9. The method, the population for use or the use according to any one of the preceding claims, wherein the checkpoint inhibitor therapy comprises two doses of 480mg Nivolumab, administered once every four weeks.

10. The method, the population for use or the use according to any one of the preceding claims, wherein the method comprises administering 5 x 10⁷ CD30-specific CAR-expressing T cells/m² to 1 x 10⁹ CD30-specific CAR-expressing T cells/m² to the subject.

11 . The method, the population for use or the use according to any one of the preceding claims, wherein the method comprises administering 1 x 10⁸ CD30-specific CAR-expressing T cells/m² to 6 x 10⁸ CD30-specific CAR-expressing T cells/m² to the subject.

12. The method, the population for use or the use according to any one of the preceding claims, wherein the method further comprises administering lymphodepleting chemotherapy to the subject, prior to administering CD30-specific CAR-T cells to the subject.

13. The method, the population for use or the use according to claim 12, wherein the lymphodepleting chemotherapy comprises administering fludarabine and bendamustine.

14. The method, the population for use or the use according to claim 13 wherein the lymphodepleting chemotherapy comprises administering fludarabine at a dose of 15 to 60 mg/m² per day, for 2 to 6 consecutive days.

15. The method, the population for use or the use according to any one of claim 13 or claim 14, wherein the method comprises administering fludarabine at a dose of 30 mg/m² per day, for 3 consecutive days.

16. The method, the population for use or the use according to any one of claims 13 to 15, wherein the method comprises administering bendamustine at a dose of 35 to 140 mg/m² per day, for 2 to 6 consecutive days.

17. The method, the population for use or the use according to any one of claims 13 to 16, wherein the method comprises administering bendamustine at a dose of 70 mg/m² per day, for 3 consecutive days.

18. The method, the population for use or the use according to any one of the preceding claims, wherein the method comprises:

(ii) administering CD30-specific CAR-expressing T cells to the subject at a dose of 2 x 10⁸ CD30- specific CAR-expressing T cells/m² to 6 x 10⁸ CD30-specific CAR-expressing T cells/m²; and 59

(iii) administering two doses of Nivolumab, wherein each dose comprises 480mg administered every four weeks. The method, the population for use or the use according to claim 18 wherein the method further includes:

(iv) after 8 weeks, administering Autologous Stem Cell Therapy to the subject. The method, the population for use or the use according to claim 18 or claim 19 wherein the method further comprises, after step (i) and before step (ii):

(a) administering fludarabine at a dose of 30 mg/m² per day and bendamustine at a dose of 70 mg/m² per day to a subject for 3 consecutive days. The method, the population for use or the use according to any one of the preceding claims, 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, HTLV-1 -associated adult T cell leukemia/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 method, the population for use or the use according to any one of the preceding claims, wherein the CD30-positive cancer is a relapsed or refractory CD30-positive cancer. The method, the population for use or the use according to any one of the preceding claims, wherein 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). The method, the population for use or the use according to claim 23, wherein the signalling domain comprises an amino acid sequence having at least 80% amino acid sequence identity to SEQ ID NO:26. The method, the population for use or the use according to claim 23 or claim 24, wherein the transmembrane domain is derived from the transmembrane domain of CD28. 60

26. The method, the population for use or the use according to any one of claims 23 to 25, wherein the transmembrane domain comprises an amino acid sequence having at least 80% amino acid sequence identity to SEQ ID NO:20.

27. The method, the population for use or the use according to any one of claims 23 to 26, 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.

28. The method, the population for use or the use according to any one of claims 23 to 27, wherein the antigen-binding domain comprises an amino acid sequence having at least 80% amino acid sequence identity to SEQ ID NO:18.

29. The method, the population for use or the use according to any one of claims 23 to 28, wherein the signalling domain comprises: (a) an amino acid sequence derived from the intracellular domain of CD3 .

30. The method, the population for use or the use according to any one of claims 23 to 29, wherein the signalling domain comprises an amino acid sequence having at least 80% amino acid sequence identity to SEQ ID NO:25.

31 . The method, the population for use or the use according to any one of claims 23 to 30, wherein the CAR additionally comprises a hinge region provided between the antigen-binding domain and the transmembrane domain.

32. The method, the population for use or the use according to any one of claims 23 to 31 , wherein the hinge region comprises an amino acid sequence having at least 80% amino acid sequence identity to SEQ ID NO:33.

33. The method, the population for use or the use according to any one of claims 23 to 32, wherein the CAR comprises an amino acid sequence having at least 80% amino acid sequence identity to SEQ ID NO:35 or 36.