Tumor-targeting PIC filaments

Abstract

The invention is in the field of immunotherapy, in particular in the field of immunotherapy to treat cancer. The invention is directed to conjugates that comprise a polyisocyanide (PIC) polymer and a tumor-targeting moiety. The conjugates can target cancer cells by binding to membrane proteins of those cancer cells, thereby promoting cell death. The invention further relates to the use of such conjugates as a medicament.

Description

[0001] Tumor-targeting PIC filaments

[0002] Field of the invention

[0003] The invention is in the field of immunotherapy, in particular in the field of immunotherapy to treat cancer. The invention is directed to conjugates that comprise a polyisocyanide (PIC) polymer and a tumor-targeting moiety. The conjugates can target cancer cells by binding to membrane proteins of those cancer cells, thereby promoting cell death. The invention further relates to the use of such conjugates as a medicament.

[0004] Background art

[0005] Haematological malignancies present a major health burden worldwide. For example, nonHodgkin lymphoma is the most common haematological malignancy, with 544,000 new diagnoses reported in 2020. Diffuse large B-cell lymphoma (DLBCL) is its most prevalent subtype and represents 30-40% of cases. Multiple myeloma (MM) also represents one of the most common haematological malignancies, with about 176,400 new cases reported in 2020. MM and DLBCL are aggressive diseases that are caused by clonal proliferation of malignant B-cells. In DLBCL, the malignant cells arise from germinal center or activated B-cells, whereas in MM, the malignant cells are terminally differentiated plasma cells, the end stage of B-cell development.

[0006] For the past 20 years, the golden standard in DLBCL treatment has been the chemoimmunotherapy R-CHOP (rituximab combined with a chemotherapeutic regimen) (Major, A. et al., Clin Adv Hematol Oncol., 2021 Nov;19(11):698— 7092021). Rituximab is a chimeric monoclonal antibody that targets CD20 (aCD20), a four-transmembrane protein with a tetraspaninlike structure that is mainly expressed on B cells. CD20 is expressed from the pre-B cell to memory B cell differentiation stages and is absent on pro-B cells and plasma cells, providing a window of opportunity for rituximab to remove aberrant B cells during their development while allowing development of new, healthy B cells and maintaining immune responses initiated by plasma cells. The standard of care in MM has been combination therapy including a proteasome inhibitor (bortezomib or carfilzomib), an immunomodulatory drug (lenalidomide or thalidomide), dexamethasone, and increasingly, an anti-CD38 monoclonal antibody daratumumab. Daratumumab is a human monoclonal antibody that targets CD38 (aCD38), a transmembrane glycoprotein highly expressed on malignant plasma cells. Because CD38 is largely absent on early B-cell progenitors and other non-plasma hematopoietic cells, daratumumab selectively targets malignant plasma cells while largely preserving normal hematopoiesis and immune function, supporting their use in first-line therapy and maintenance regimens.

[0007] Current treatment strategies for haematological malignancies, like non-Hodgkin lymphoma and MM are suboptimal. For example, a disadvantage of the R-CHOP regimen is that 30-50% of DLBCL patients is not cured. Such R-CHOP failures are principally due to either primary refractoriness or relapse after reaching complete remission (Coiffier , B. Sarkozy, C., 2016, doi: 10.1182 / asheducation-2016.1.366). A survival time of only 6.3 months has been reported after resistance to R-CHOP. Similarly, about 15-20% of newly diagnosed MM patients relapsed orprogressed by four years despite having received the standard of care MM treatment (Sonneveld, P. et al, N Engl J Med., 2024 Jan 25;390(4):301-313). In addition, MM has not historically been treated when in its "smoldering" stage, since the drugs utilized will then be of diminished efficacy if the disease progresses to a symptomatic stage. Thus, the standard of care comprises "watchful waiting" while the disease smoldered.

[0008] Polyisocyanide (PIC) polymers form a stiff helical backbone. Hydrogen bonds between alanines in the side chains stabilize the helical structure, providing the polymer with a semiflexibility that improves its interaction with cells. Previous studies have functionalized PICs with peptide major histocompatibility complexes and costimulatory molecules, such as IL-2 and anti-CD28 antibodies, to drive the activation and expansion of antigen-specific T cells (Weiss, L., et al., 2023, doi:10.1021 / acsnano.2c11884). Such T cells migrate to the site of the tumor following their activation, and subsequently recognize and kill the tumor cells.

[0009] There is a need for more effective treatment strategies for cancer patients. There is an ongoing need in the art for achieving more efficient tumor cell killing. There is a need to enhance tumor cell cytotoxicity.

[0010] Summary of the invention

[0011] The conjugates according to the present invention target tumor cells directly, independently of T cell activation and migration. This enables a more straight-forward way to delay and / or inhibit tumor growth. The objective of the present invention is to provide more effective treatment strategies for cancer patients, such as DLBCL patients, by establishing enhanced tumor cell cytotoxicity. The invention achieves this by providing a conjugate of a PIC polymer and binding moieties such as antibodies that target membrane proteins of cancer cells. Exemplary antibodies target CD20 (such as rituximab) and / or CD37 (such as otlertuzumab), and / or CD38 (such as daratumumab and isatuximab). The invention demonstrates that multivalent antibody presentation can be achieved by attaching antibodies that target membrane proteins on tumor cells, such as CD20, CD37, and CD38, to PIC polymers. The inventors surprisingly found an upregulation of the rituximab-associated apoptotic markers p-p38 and intracellular Ca2+after treatment with aCD20 attached to a PIC polymer in comparison to soluble aCD20. Furthermore, the inventors surprisingly found that aCD38 attached to a PIC polymer also increased apoptotic markers, such as cleaved caspase-3 and intracellular Ca2+levels, compared to their soluble aCD38. In line with that, the inventors found that tumor cell death induced by tumor-targeting antibodies was enhanced upon attaching the tumor-targeting antibodies to a PIC polymer.

[0012] The invention provides a conjugate comprising a PIC polymer and a first binding moiety capable of binding a membrane protein of a cancer cell. Preferably, the binding moiety is an antibody or a fragment thereof. Preferably, the number of first binding moieties per PIC polymer is between 1 to 10, preferably between 1 to 4, more preferably it is 2 or 3. Preferably, the PIC polymer has a length of about 100 to 1000 nm, preferably about 200 to 600 nm, more preferably about 400 nm. Preferably, the cancer cell is a haematological cancer cell, preferably a lymphoma cell, a chronic lymphocytic leukemia cell, or a multiple myeloma cell, more preferably a non-Hodgkinlymphoma cell. In some embodiments, the haematological cancer cell is a lymphoma cell, a leukemia cell, or a myeloma cell, preferably a lymphoma cell, a chronic lymphocytic leukemia cell, or a multiple myeloma cell.

[0013] In some embodiments, the first binding moiety is capable of binding CD20, CD38, CD45, CD5, CD19, CD10, CD30, CD7, CD2, CD23, CD43, CD103, CD13, CD33, CD1a or CD22, preferably CD20, CD38, CD45, CD5, CD19, or CD10, more preferably CD20 or CD38, most preferably CD20. Preferably, the first binding moiety is Ritixumab, Daratumumab, Isatuximab, Brentuximab vedotin, Epcoritamab, Glofitamab, Ibritumomab tiuxetan, Mogamulizumab, Obinutuzumab, Ocrelizumab, Ofatumumab, Polatuzumab vedotin, Ripertamab, Ublituximab, or Zuberitamab, preferably Ritixumab, Daratumumab, Isatuximab, Ibritumomab tiuxetan, Obinutuzumab, Ocrelizumab, Ofatumumab, Ripertamab, Ublituximab, or Zuberitamab, more preferably Ritixumab, Ibritumomab tiuxetan, Obinutuzumab, or Ofatumumab, most preferably Rituximab.

[0014] In some embodiments, the first binding moiety is capable of binding CD20, wherein the first binding moiety is preferably Rituximab, Ibritumomab tiuxetan, Obinutuzumab, Ocrelizumab, Ofatumumab, Ublituximab, Zuberitamab, Veltuzumab, Ocaratuzumab, or Tositumomab, preferably Rituximab, Ibritumomab tiuxetan, Obinutuzumab, Ocrelizumab, Ofatumumab, or Ublituximab, Zuberitamab, preferably it is Rituximab. Preferably, the number of first binding moieties per polyisocyanide polymer is between 1 to 10, preferably between 1 to 4, more preferably it is 2 or 3, in conjugates comprising a first binding moiety capable of binding CD20.

[0015] In some embodiments, the first binding moiety is capable of binding a membrane protein of the tetraspanin superfamily, preferably CD37, CD9, CD53, CD63, CD81 , TSPAN13, TSPAN4, CD151 , or CD82, most preferably CD37. Preferably, the first binding moiety is Otlertuzumab, Lilotomab, or Naratuximab, preferably Otlertuzumab. In other preferred embodiments, the first binding moiety is Naratuximab.

[0016] In some embodiments, the first binding moiety is capable of binding CD38, wherein the first binding moiety is preferably Daratumumab, Isatuximab, Felzartamab, Mezagitamab, HLX15, or CID-103, preferably Daratumumab, Isatuximab, Felzartamab, or Mezagitamab, more preferably Daratumumab or Isatuximab. In some embodiments, the first binding moiety is Daratumumab. In some embodiments, the first binding moiety is Isatuximab. Preferably, the number of first binding moieties per polyisocyanide polymer is between 1 to 20, preferably between 3 to 10, more preferably between 5-9, most preferably between 6-8, in conjugates comprising a first binding moiety capable of binding CD38.

[0017] Also provided is a conjugate further comprising a second binding moiety capable of binding a membrane protein of a cancer cell. Preferably, the total number of first and second binding moieties per PIC polymer is between 2 to 10, preferably between 2 to 4, more preferably it is 2 or 3. Preferably, the conjugate comprises at least one first binding moiety and at least one second binding moiety, wherein the first binding moiety is capable of binding CD20, CD38, CD45, CD5, CD19, CD10, CD30, CD7, CD2, CD23, CD43, CD103, CD13, CD33, CD1a or CD22, preferably CD20, CD38, CD45, CD5, CD19, or CD10, more preferably CD20 or CD38, most preferably CD20,wherein the second binding moiety is capable of binding a membrane protein of the tetraspanin superfamily, preferably CD37, CD9, CD53, CD63, TSPAN13, TSPAN4, CD151 , CD81 , or CD82, most preferably CD37.

[0018] Also provided is a conjugate for use as a medicament, wherein the medicament is preferably for treating cancer, more preferably haematological cancer such as lymphoma, chronic lymphocytic leukemia, or multiple myeloma, even more preferably non-Hodgkin lymphoma, most preferably diffuse large B cell lymphoma. In some embodiments, the medicament is for treating lymphoma, leukemia, or myeloma, most preferably lymphoma, chronic lymphocytic leukemia, or multiple myeloma. Preferably, the conjugate is for promoting p38 phosphorylation in the cancer cell. Preferably, the conjugate is for promoting p38 phosphorylation and / or calcium influx in the cancer cell.

[0019] Also provided is a method for treating cancer in a subject, the method comprising the step of administering the conjugate to the subject.

[0020] Detailed description

[0021] The invention provides conjugates that comprise a PIC polymer and a tumor-targeting moiety. The conjugates of the invention are capable of targeting cancer cells by binding to one or more membrane proteins of said cancer cells, thereby promoting cell death of said cancer cells. Accordingly, the invention provides a conjugate comprising a PIC polymer and a first binding moiety capable of binding a membrane protein of a cancer cell. The inventors surprisingly found an upregulation of apoptotic markers after treatment with such conjugates.

[0022] Polyisocyanide (PIC) polymer

[0023] As used herein, a PIC polymer is formed from polymerised isocyanopeptide monomers, to which molecules such as a binding moiety can be attached. Such attachment can be via a linker or sidechain, such as a PEG sidechain, extending from the backbone of the PIC polymer and separating the backbone monomers from the attached molecules. “Attached” as used herein is taken to mean the components are chemically bonded (e.g. covalently bonded). In some embodiments, the PIC polymer is obtainable by a method comprising nickel-catalysed polymerization of a mixture comprising methoxy-terminated and azide- terminated isocyanopeptide monomers.

[0024] A PIC polymer has a worm-like (i.e. filamentous) structure arising from their helical form and does not form a random coil when in an aqueous medium, and allows for effective binding of an attached binding moiety to a membrane protein on a cancer cell. A PIC polymer conjugated to binding moieties can also be referred to as a PIC nanofilament or a PIC immunofilament (IF). Suitable PIC polymers may be synthesised according to methods known in the art by polymerisation of isocyanopeptide monomers (see for example Mandal, S., et al., 2013 (doi:10.1039 / c3sc51399h), and Koepf, M. et al., 2013 (doi: 10.1016 / j.eurpolymj.2013.01.009).

[0025] In some embodiments, the PIC polymer can be prepared from isocyanide monomers that have been derived from amino acids or peptides. Techniques for converting amino acids intoisocyanopeptides suitable for use as monomers in the PIC polymers are known in the art. When an isocyanopeptide monomer is derived from an amino acid, it may be derived from any suitable naturally-occurring or non-naturally-occurring amino acid, and may for example be a D- or L- amino acid (or may have an R-confirmation or an S-confirmation, referring to the chiral alpha carbon). Selection of a suitable isomer would be familiar to the skilled person and may be determined, for example, by the intended “handedness” of the PIC polymer, which is helical in structure. Preferred monomers are as shown below, wherein the chiral centre adjacent to the isocyanide moiety is preferably R and the chiral centre adjacent to the oligo(ethylene glycol) is preferably S.

[0026]

[0027] The PIC polymer may or may not be biologically degradable (and, in particular, degradable in the human or animal body).

[0028] In some embodiments, the PIC polymer is formed by polymerisation of a nonfunctional monomer and functional monomer. In this context, a functional monomer is a monomer having a functional moiety suitable for use in an attachment reaction, such as click chemistry. Binding moieties can then be attached as a result of an attachment reaction with the functional moiety. Suitable means for attaching molecules to the polymeric backbone are familiar to the skilled person. For example, when attachment is via a click reaction, the binding moiety to be attached and functional moiety form an appropriate click pair. Suitable click pairs would be familiar to the skilled person and include azide and a bicyclononyne (BCN), dibenzocyclooctyne (DBCO), or dibenzoazacyclooctyne (DI BO) group, a copper(l)-catalyzed azide alkyne cycloaddition (CuAAC) pair, or tetrazine and transcyclooctene (TOO).

[0029] In some embodiments, the functional monomer is functionalised with a suitable functional moiety such as a Michael acceptor, and active ester, or a clickable moiety, for example azide, N-hydroxysuccinimide (NHS), vinyl sulfone, acetylene, NH2, COOH, maleimide, tetrazine, thiol or transcyclooctene (TOO). In preferred embodiments, the functionalised monomer is functionalised with an otherwise inert moiety such as an azide moiety. The non-functional monomer is not functionalised with such a group. In some embodiments, the polymer is obtainable by polymerisation of a methoxy-terminated non-functional monomer and a functional, preferably an azide-terminated functional monomer. For legibility, a backbone formed by this method is referred to as a polymer, rather than co-polymer, despite being formed of different monomers.

[0030] In some embodiments, the ratio of functionalised to non-functionalised monomer is selected such that the reaction produces polymers having a functional group (for example an azide group) every 1-10 nm, preferably every 1.5-8, more preferably every 2-5 nm, even more preferably every 2.5-4 nm, such as every 3-3.5 nm. In certain embodiments the ratio is chosen to produce a polymer having a functional group (preferably an azide group) every 3.5 nm. It will be appreciated by theskilled person that such polymerisation reactions are random and therefore the described separation of functional groups refers to the statistical or mean separation distance across the synthesised polymer sample, and allows for individually selected functional groups within a PIC polymer to be positioned at greater or smaller distances without the PIC polymer falling outside the limits of the feature. In general, it can be seen as the length of the polymer divided by the number of functional groups. In some such embodiments, the ratio of non-functional monomer (y) to functional monomer (x) is in the range of from 100:1 to 10:1 , optionally 50:1 to 10:1 , such as 45:1 to 15:1 , preferably 40:1 to 20:1 , more preferably 35:1 to 25:1 , most preferably about 30:1.

[0031] The number of monomer units “n” in the PIC polymer is determined by the length of the PIC polymers in accordance with the embodiments described herein. In some embodiments, “n” is in the range of from 1000 to 20000. In certain preferred embodiments, “n” is in the range of from 1000 to 10,000. In certain preferred embodiments, “n” is in the range of from 2000 to 10,000. In certain preferred embodiments, “n” is in the range of from 3000 to 5000, for example about 4000.

[0032] In some embodiments, a PIC polymer is nano-sized - that is, typically has a length measurable on a nanoscale of from 1 to 1000 nm, preferably 25 to 1000 nm. The conjugates provided herein are particularly effective at inducing cancer cell cytotoxicity because the length of the PIC polymer allows for presentation of the attached molecules at scales similar to that of the receptor spacing on the cells. Accordingly, in some embodiments, the PIC polymer has a length of about 1 to 1000 nm, such as about 10 to 900 nm, preferably about 50 to 800 nm, more preferably about 100 to 700 nm, most preferably about 200 to 600 nm, such as about 300 to 500 nm or about 400 nm. In some embodiments, In some embodiments, the PIC polymer has a length of about 100 to 1000 nm, preferably about 200 to 600 nm, more preferably about 400 nm. It will be appreciated by the skilled person that, when synthesising a PIC polymer or co-polymer, a statistical distribution of PIC polymer lengths is obtained. Therefore, where, say, a composition includes a large number of PIC polymers, the embodiments of length described above refers to the statistical length of the PIC polymer sample, for example the mean length of the PIC polymer.

[0033] In some embodiments, the PIC polymer has a persistence length of from 1 % to 20% of contour length, preferably of from 2% to 15% of contour length. In some embodiments, the PIC polymer has a persistence length of from 5% to 15% of contour length, preferably from 8% to 12%, most preferably about 10%. In certain preferred embodiments, the PIC polymer is having a persistence length in the range of from 1 order of magnitude lower than the contour length to the same order of magnitude of the contour length. PIC polymers having a persistence length in these ranges are especially capable of dynamic rearrangement without coiling into a polymer ball. The values described refer to persistence length in water, and means for determining the persistence length would be familiar to the skilled person.

[0034] Binding moiety

[0035] The term “binding moiety” refers to molecules capable of binding to another molecule or complex, and is known to the skilled person. In some embodiments, a binding moiety is an antibody or a fragment thereof, such as a monoclonal antibody. In some embodiments, a binding moiety isan immunoglobulin-derived binding moiety, e.g. comprising both an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). Examples of such binding moieties include single-chain Fv (scFv), single-chain antibody, Fv, single-chain Fv2 (scFv2), Fab, and Fab'. In some embodiments, a binding moiety is an immunoglobulin-derived binding moiety from a single domain antibody consisting only of heavy chains and devoid of light chains as are known e.g. from camelids, wherein the antigen-binding site is present on, and formed by, the single variable domain (also referred to as an "immunoglobulin single variable domain" or "ISVD"). Examples of such ISVDs include the single variable domains of camelid heavy chain antibodies (VHHs), also referred to as nanobodies, domain antibodies (dAbs), and single domains derived from shark antibodies (IgNAR domains). In other embodiments, a binding moiety comprises a non-immunoglobulin-derived domain capable of specifically binding to an antigen or epitope, such as DARPpins; Affilins; anticalins, etc.

[0036] The term "antibody" herein is used in the broadest sense and specifically includes full-length monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g. bispecific antibodies), and antibody fragments and derivatives, so long as they exhibit the desired biological and / or immunological activity. Various techniques relevant to the production of antibodies are provided in, e.g., Harlow, E and Lane, D., 1988 (“Antibodies: A Laboratory Manual”, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). An antibody can be human and / or humanized. "Humanized" forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody.

[0037] The term "monoclonal antibody" as used herein is not limited to antibodies produced through hybridoma technology. The term "monoclonal antibody" refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced. Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example in Harlow, E and Lane, D., 1988 (Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) and Hammerling, G. J. et al., 1981 ("Monoclonal Antibodies and T-Cell Hybridomas," Elsevier, N.Y., pp. 563-681).

[0038] In some embodiments, a binding moiety is a monoclonal antibody for use in treating cancer, such as 3F8, Abagovomab, Abituzumab, Adecatumumab, Alacizumab pegol, Alemtuzumab, Altumomab pentetate, Amatuximab, Amivantamab, Anatumomab mafenatox, Andecaliximab[, Anetumab ravtansine, Apolizumab, Arcitumomab, Ascrinvacumab, Atezolizumab, Avelumab, Azintuxizumab vedotin, Bavituximab, BCD-100, Bectumomab, Belantamab mafodotin, Bemarituzumab, Bermekimab, Bevacizumab, Bivatuzumab, Blinatumomab, Blontuvetmab, Brentuximab vedotin, Brontictuzumab, Cabiralizumab, Camidanlumab tesirine, Camrelizumab, Cantuzumab mertansine, Cantuzumab ravtansine, Catumaxomab, cBR96-doxorubicin immunoconjugate, Cemiplimab, Cergutuzumab amunaleukin, Cetrelimab, Cetuximab, Cibisatamab, Cirmtuzumab, Citatuzumab bogatox, Cixutumumab, Clivatuzumab tetraxetan,Codrituzumab, Cofetuzumab pelidotin, Coltuximab ravtansine, Conatumumab, Cosibelimab, Cusatuzumab, Dacetuzumab, Dalotuzumab, Dapirolizumab pegol, Daratumumab, Demcizumab, Denintuzumab mafodotin, Denosumab, Depatuxizumab mafodotin, Derlotuximab biotin, Detumomab, Dinutuximab, Dinutuximab beta, Divozilimab, Dostarlimab, Drozitumab, Duligotuzumab, Durvalumab, Dusigitumab, Duvortuxizumab, Ecromeximab, Edrecolomab, Elgemtumab, Elotuzumab, Ektomab, Emactuzumab, Emibetuzumab, Enapotamab vedotin, Enavatuzumab, Enfortumab vedotin, Enoblituzumab, Ensituximab, Epcoritamab, Epratuzumab, Ertumaxoma, Etaracizumab, Farletuzumab, FBTA05, Ficlatuzumab, Figitumumab, Flanvotumab, Flotetuzumab, Fresolimumab, Futuximab, Galiximab, Gancotamab, Ganitumab, Gatipotuzumab, Gemtuzumab ozogamicin, Girentuximab, Glembatumumab vedotin, Glofitamab, Sintilimab, Ibritumomab tiuxetan, Icrucumab, Ifabotuzumab, Igovomab, lladatuzumab vedotin, Imalumab, Imaprelimab, Imgatuzumab, Indatuximab ravtansine, Indusatumab vedotin, Inebilizumab, Intetumumab, Inotuzumab ozogamicin, Ipilimumab, Iratumumab, Isatuximab, Istiratumab, KappaMAB, Labetuzumab, Lacnotuzumab, Ladiratuzumab vedotin, Lebrikizumab, Lenzilumab, Leronlimab, Lexatumumab, Lifastuzumab vedotin, Loncastuximabtesirine, Losatuxizumab vedotin, Lilotomab satetraxetan, Lintuzumab, Lirilumab, Lorvotuzumab mertansine, Lucatumumab, Lumiliximab, Lumretuzumab, Mapatumumab, Margetuximab, Matuzumab, Milatuzumab, Mirvetuximab soravtansine, Mitumomab, Modotuximab, Mogamulizumab, Monalizumab, Mosunetuzumab, Moxetumomab pasudotox, Nacolomab tafenatox, Naptumomab estafenatox, Naratuximab, emtansine, Narnatumab, Navicixizumab, Necitumumab, Nesvacumab, Nimotuzumab, Nivolumab, Nofetumomab merpentan, Obinutuzumab, Ocaratuzumab, Ofatumumab, Olaratumab, Oleclumab, Omburtamab, Onartuzumab, Ontuxizumab , Oportuzumab monatox, Oregovomab, Otlertuzumab, Pamrevlumab, Panitumumab, Pankomab, Parsatuzumab, Pasotuxizumab, Patritumab, PDR001, Pembrolizumab, Pemtumomab, Pertuzumab, Pidilizumab, Pinatuzumab vedotin, Polatuzumab vedotin, Pritumumab, Racotumomab, Radretumab, Ramucirumab, Ranibizumab, Relatlimab, Retifanlimab, Rilotumumab, Rituximab, Robatumumab, Rosmantuzumab, Rovalpituzumab tesirine, Sacituzumab govitecan, Samalizumab, Samrotamab vedotin, Satumomab pendetide, Seribantumab, Sibrotuzumab, Siltuximab, Sirtratumab vedotin, Sofituzumab vedotin, Solitomab, Spartalizumab, Sugemalimab, Tabalumab, Tacatuzumab tetraxetan, Tafasitamab, Talacotuzumab, Talquetamab, Taplitumomab paptox, Tarextumab, Tarlatamab, Tavolimab, Teclistamab, Telisotuzumab, Telisotuzumab vedotin, Tenatumomab, Tepoditamab, Tetulomab, Tigatuzumab, Timigutuzumab, Tiragotumab, Tislelizumab, Tisotumab vedotin, Tomuzotuximab, Tositumomab, Tovetumab, Trastuzumab, Trastuzumab duocarmazine, Trastuzumab emtansine, Tremelimumab, Tucotuzumab celmoleukin, Ublituximab, Ulocuplumab, Urelumab, Utomilumab, Vadastuximab talirine, Vandortuzumab vedotin, Vantictumab, Vanucizumab, Varlilumab, Veltuzumab, Vesencumab, Volociximab, Vonlerolizumab, Vorsetuzumab mafodotin, Votumumab, XMAB-5574, Zalutumumab, Zanidatamab, Zanolimumab, Zatuximab, Zenocutuzumab, Ziralimumab, and Zolbetuximab. Preferably, the monoclonal antibody is for use in treating a haematological cancer, such as Alemtuzumab, Apolizumab, Bectumomab, Belantamab mafodotin, Blinatumomab, Blontuvetmab, Brentuximab vedotin, Brontictuzumab,Camidanlumab tesirine, Cirmtuzumab, Coltuximab ravtansine, Dacetuzumab, Daratumumab, Denintuzumab mafodotin, Detumomab, Divozilimab, Dusigitumab, Elotuzumab, Epcoritamab, FBTA05, Flotetuzumab, Galiximab, Gemtuzumab ozogamicin, Glofitamab, Ibritumomab tiuxetan, Inebilizumab, Inotuzumab ozogamicin, Iratumumab, Isatuximab, KappaMAB, Lebrikizumab, Lenzilumab, Loncastuximab tesirine, Lirilumab, Lucatumumab, Lumiliximab, Lumretuzumab, Mapatumumab, Margetuximab, Milatuzumab, Mogamulizumab, Mosunetuzumab, Moxetumomab pasudotox, Obinutuzumab, Ofatumumab, Otlertuzumab, Pinatuzumab vedotin, Polatuzumab vedotin, Rituximab, Tabalumab, Tafasitamab, Talacotuzumab, Talquetamab, Taplitumomab paptox, Teclistamab, Tetulomab, Tositumomab, Ublituximab, Ulocuplumab, Utomilumab, Vadastuximab talirine, Varlilumab, Veltuzumab and XMAB-5574, more preferably, Alemtuzumab, Blinatumomab, Brentuximab vedotin, Daratumumab, Elotuzumab, Gemtuzumab ozogamicin, Ibritumomab tiuxetan, Inotuzumab ozogamicin, Mogamulizumab, Moxetumomab pasudotox, Obinutuzumab, Ofatumumab, Polatuzumab vedotin, Rituximab, Teclistamab, Tositumomab.

[0039] In some embodiments, the first binding moiety is Ritixumab, Daratumumab, Isatuximab, Brentuximab vedotin, Epcoritamab, Glofitamab, Ibritumomab tiuxetan, Mogamulizumab, Obinutuzumab, Ocrelizumab, Ofatumumab, Polatuzumab vedotin, Ripertamab, Ublituximab, or Zuberitamab, preferably Ritixumab, Daratumumab, Isatuximab, Ibritumomab tiuxetan, Obinutuzumab, Ocrelizumab, Ofatumumab, Ripertamab, Ublituximab, or Zuberitamab, more preferably Ritixumab, Ibritumomab tiuxetan, Obinutuzumab, or Ofatumumab, most preferably Rituximab.

[0040] In some embodiments, a binding moiety is a monoclonal antibody capable of binding CD20. Preferably, the monoclonal antibody capable of binding CD20 is Ibritumomab tiuxetan, Epcoritamab, Glofitamab, Obinutuzumab, Ocrelizumab, Ofatumumab, Ripertamab, Ritixumab, Ublituximab or Zuberitamab, preferably Ibritumomab tiuxetan, Obinutuzumab, Ofatumumab or Ritxumab, more preferably Rituxumab. In some embodiments, a binding moiety is a monoclonal antibody for use in treating non-Hodgkin lymphoma, such as Rituximab, Obinutuzumab, Polatuzumab vedotin, Mogamulizumab, Brentuximab vedotin, Glofitamab and Epcoritamab.

[0041] In some embodiments, a binding moiety is a monoclonal antibody capable of binding CD38, such as Daratumumab, Isatuximab, Felzartamab, Mezagitamab, HLX15, or CID-103, preferably Daratumumab, Isatuximab, Felzartamab, or Mezagitamab, more preferably Daratumumab or Isatuximab. In some embodiments, the binding moiety is Daratumumab. In some embodiments, the binding moiety is Isatuximab. Accordingly, in preferred embodiments, a binding moiety is a monoclonal antibody capable of binding CD38, such as Daratumumab or Isatuximab.

[0042] In some embodiments, a binding moiety is a monoclonal antibody capable of binding CD37, such as Naratuximab, Lilotomab, Bl 836826 and Otlertuzumab. In some embodiments, the first binding moiety is Otlertuzumab, Lilotomab, or Naratuximab, preferably Otlertuzumab. In some embodiments, the first binding moiety is Naratuximab.

[0043] As used herein, “capable of binding” means that a binding moiety can facilitate a physical interaction between a membrane protein on a cancer cell and the conjugate of the invention that comprises said binding moiety.As used herein “a membrane protein” is given its customary meaning, and thus generally refers to proteins that are part of or interact with a cell membrane. A membrane protein can be an integral membrane protein (i.e. a permanent part of a membrane), a transmembrane membrane protein (i.e. penetrating a membrane), an integral monotopic membrane protein, (i.e. associated with one or the other side of a membrane), and / or a peripheral membrane protein (i.e. transiently associated with a membrane). A membrane protein can for example be a membrane receptor, a transport protein, a membrane enzyme, and / or a cell adhesion molecule. In some embodiments, the membrane protein is absent on pro-B cells and plasma cells.

[0044] In some embodiments, the membrane protein is CD20, CD38, CD45, CD5, CD19, CD10, CD30, CD7, CD2, CD23, CD43, CD103, CD13, CD33, CD1a or CD22, preferably CD20, CD38, CD45, CD5, CD19, or CD10, more preferably CD20 or CD38, most preferably CD20.

[0045] In some embodiments, the membrane protein is a membrane protein of the tetraspanin superfamily, such as TSPAN1 (i.e. TSP-1), TSPAN2 (i.e. TSP-2), TSPAN3 (i.e. TSP-3), TSPAN4 (i.e. TSP-4, NAG-2), TSPAN5 (i.e. TSP-5), TSPAN6 (i.e. TSP-6), TSPAN7 (i.e. CD231 , TALLA-1), TSPAN8 (i.e. CO-029), TSPAN9 (i.e. NET-5), TSPAN10 (i.e. oculospanin), TSPAN11 (i.e. CD151-like), TSPAN12 (i.e. NET-2), TSPAN13 (i.e. NET-6), TSPAN14, TSPAN15 (i.e. NET-7), TSPAN16 (i.e. TM4-B), TSPAN17, TSPAN18, TSPAN19, TSPAN20 (i.e. UP1b, UPK1 B), TSPAN21 (i.e. UP1a, UPK1A), TSPAN22 (i.e. RDS, PRPH2), TSPAN23 (i.e. ROM1), TSPAN24 (i.e. CD151), TSPAN25 (i.e. CD53), TSPAN26 (i.e. CD37), TSPAN27 (i.e. CD82), TSPAN28 (i.e. CD81), TSPAN29 (i.e. CD9), TSPAN30 (i.e. CD63), TSPAN31 (i.e. SAS), TSPAN32 (i.e. TSSC6) and TSPAN33. Preferably, the first binding moiety is capable of binding a membrane protein of the tetraspanin superfamily, preferably CD37, CD9, CD53, CD63, CD81 , TSPAN13, TSPAN4, CD151 , or CD82, most preferably CD37 CD37, CD9, CD53, CD63, CD81 , TSPAN13, TSPAN4, CD151 , or CD82, most preferably CD37.

[0046] In some embodiments, the first binding moiety is capable of binding CD20, CD38, CD45, CD5, CD19, CD10, CD30, CD7, CD2, CD23, CD43, CD103, CD13, CD33, CD1a or CD22, preferably CD20, CD38, CD45, CD5, CD19, or CD10, more preferably CD20 or CD38, most preferably CD20

[0047] In some embodiments, the first binding moiety is capable of binding a membrane protein of the tetraspanin superfamily, preferably CD37, CD9, CD53, CD63, CD81 , TSPAN13, TSPAN4, CD151 , or CD82, most preferably CD37.

[0048] Conjugate

[0049] Conjugates according to the invention comprise a PIC polymer and a first binding moiety capable of binding a membrane protein of a cancer cell. A conjugate is a compound formed by joining two or more different molecules, for example as described throughout the application and reduced to practice in the examples section. Preferred conjugates comprise a first binding moiety and a second binding moiety. For example the first binding moiety can bind one target, and the second binding moiety can bind another target. In preferred embodiments the first binding moiety and the second binding moiety are different binding moieties, more preferably capable of bindingdifferent membrane proteins of a cancer cell.

[0050] In some embodiments, the conjugate comprises a plurality of first binding moieties, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 or 20. In some embodiments, the number of first binding moieties per PIC polymer is between 1 to 10, preferably between 1 to 4, more preferably it is 2 or 3. In some embodiments, the number of first binding moieties per PIC polymer is between 1 to 20, preferably between 2 to 15, more preferably between 3 to 12, even more preferably between 4 to 10, most preferably between 6 to 8.

[0051] In some embodiments, the conjugate comprises a first binding moiety, wherein the first binding moiety are capable of binding CD20. Preferably, the number of first binding moieties capable of binding CD20 per PIC polymer is between 1 to 10, preferably between 2 to 8, more preferably between 3 to 6. In preferred embodiments, the number of first binding moieties capable of binding CD20 per PIC polymer is at least 1 , preferably at least 2, more preferably at least 3. In preferred embodiments, the number of first binding moieties capable of binding CD20 per PIC polymer is at most 10, preferably at most 8, more preferably at most 6, most preferably at most 4. In some embodiments, the number of first binding moieties capable of binding CD20 per PIC polymer is between 1-4, preferably between 2-3, wherein the first binding moiety capable of binding CD20 is rituximab.

[0052] In some embodiments, the conjugate comprises a first binding moiety, wherein the first binding moiety is capable of binding CD38. Preferably, the number of first binding moieties capable of binding CD38 per PIC polymer is between 1 to 20, preferably between 2 to 15, more preferably between 3 to 10, even more preferably between 4 to 9, most preferably between 6 to 8. In preferred embodiments, the number of first binding moieties capable of binding CD38 per PIC polymer is at least 1 , preferably at least 2, more preferably at least 3, even more preferably at least 4, still more preferably at least 5, most preferably at least 6. In preferred embodiments, the number of first binding moieties capable of binding CD38 per PIC polymer is at most 20, preferably at most 16, more preferably at most 12, most preferably at most 8. In some embodiments, the number of first binding moieties capable of binding CD38 per PIC polymer is between 4-10, preferably between 5-9, wherein the first binding moiety capable of binding CD38 comprises daratumumab and / or isatuximab. In embodiments wherein the first binding moiety capable of binding CD38 is daratumumab, the number of first binding moieties capable of binding CD38 per PIC polymer is preferably between 4-7, more preferably between 5-6. In embodiments wherein the first binding moiety capable of binding CD38 is isatuximab, the number of first binding moieties capable of binding CD38 per PIC polymer is preferably between 7-10, more preferably between 8-9.

[0053] In some embodiments, the conjugate comprises a second binding moiety, preferably a plurality of second binding moieties, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 or 20. In some embodiments, the number of second binding moieties per PIC polymer is between 1 to 10, preferably between 1 to 4, more preferably it is 2 or 3.

[0054] In some embodiments, the conjugate is further comprising a second binding moiety capable of binding a membrane protein of a cancer cell. The total number of first and second binding moieties per PIC polymer can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 or 20.Preferably, the total number of first and second binding moieties per PIC polymer is between 2 to 10, preferably between 2 to 4, more preferably it is 2 or 3.

[0055] In some embodiments, the conjugate comprises at least one first binding moiety and at least one second binding moiety, wherein the first binding moiety is capable of binding CD20, CD38, CD45, CD5, CD19, CD10, CD30, CD7, CD2, CD23, CD43, CD103, CD13, CD33, CD1a or CD22, preferably CD20, CD38, CD45, CD5, CD19, or CD10, more preferably CD20 or CD38, most preferably CD20, and wherein the second binding moiety is capable of binding a membrane protein of the tetraspanin superfamily, preferably CD37, CD9, CD53, CD63, TSPAN13, TSPAN4, CD151 , CD81 , or CD82, most preferably CD37.

[0056] In some embodiments, the conjugate comprises at least one first binding moiety and at least one second binding moiety, wherein the first binding moiety is capable of binding CD38, and wherein the second binding moiety is capable of binding a membrane protein of the tetraspanin superfamily, preferably CD37, CD9, CD53, CD63, TSPAN13, TSPAN4, CD151 , CD81 , or CD82, most preferably CD37.

[0057] In some embodiments, the conjugate comprises at least one first binding moiety and at least one second binding moiety, wherein the first binding moiety is capable of binding CD20, and wherein the second binding moiety is capable of binding a membrane protein of the tetraspanin superfamily, preferably CD37, CD9, CD53, CD63, TSPAN13, TSPAN4, CD151 , CD81 , or CD82, most preferably CD37. In preferred embodiments, the conjugate comprises at least one first binding moiety and at least one second binding moiety, wherein the first binding moiety is capable of binding CD20, and wherein the second binding moiety is capable of binding CD37. In some embodiments, the conjugate comprises one first binding moiety and one second binding moiety, wherein the first binding moiety is capable of binding CD20, and wherein the second binding moiety is capable of binding CD37. In some embodiments, the conjugate comprises at least two first binding moieties and at least one second binding moiety, wherein the first binding moiety is capable of binding CD20, and wherein the second binding moiety is capable of binding CD37. In some embodiments, the conjugate comprises at least one first binding moiety and at least two second binding moieties, wherein the first binding moiety is capable of binding CD20, and wherein the second binding moiety is capable of binding CD37. In some embodiments, the conjugate comprises at least two first binding moieties and at least two second binding moieties, wherein the first binding moiety is capable of binding CD20, and wherein the second binding moiety is capable of binding CD37.

[0058] In some embodiments, the conjugate comprises at least one first binding moiety and at least one second binding moiety, wherein the first binding moiety is capable of binding CD22, and wherein the second binding moiety is capable of binding a membrane protein of the tetraspanin superfamily, preferably CD37, CD9, CD53, CD63, TSPAN13, TSPAN4, CD151 , CD81 , or CD82, most preferably CD37. In preferred embodiments, the conjugate comprises at least one first binding moiety and at least one second binding moiety, wherein the first binding moiety is capable of binding CD22, and wherein the second binding moiety is capable of binding CD37. In some embodiments, the conjugate comprises one first binding moiety and one second binding moiety, wherein the first binding moiety is capable of binding CD22, and wherein the second binding moiety is capable ofbinding CD37. In some embodiments, the conjugate comprises at least two first binding moieties and at least one second binding moiety, wherein the first binding moiety is capable of binding CD22, and wherein the second binding moiety is capable of binding CD37. In some embodiments, the conjugate comprises at least one first binding moiety and at least two second binding moieties, wherein the first binding moiety is capable of binding CD22, and wherein the second binding moiety is capable of binding CD37. In some embodiments, the conjugate comprises at least two first binding moieties and at least two second binding moieties, wherein the first binding moiety is capable of binding CD22, and wherein the second binding moiety is capable of binding CD37.

[0059] In some embodiments, the conjugate comprises at least one first binding moiety and at least one second binding moiety, wherein the first binding moiety is capable of binding CD20, and wherein the second binding moiety is capable of binding CD22. In preferred embodiments, the conjugate comprises at least one first binding moiety and at least one second binding moiety, wherein the first binding moiety is capable of binding CD20, and wherein the second binding moiety is capable of binding CD22. In some embodiments, the conjugate comprises one first binding moiety and one second binding moiety, wherein the first binding moiety is capable of binding CD20, and wherein the second binding moiety is capable of binding CD22. In some embodiments, the conjugate comprises at least two first binding moieties and at least one second binding moiety, wherein the first binding moiety is capable of binding CD20, and wherein the second binding moiety is capable of binding CD22. In some embodiments, the conjugate comprises at least one first binding moiety and at least two second binding moieties, wherein the first binding moiety is capable of binding CD20, and wherein the second binding moiety is capable of binding CD22. In some embodiments, the conjugate comprises at least two first binding moieties and at least two second binding moieties, wherein the first binding moiety is capable of binding CD20, and wherein the second binding moiety is capable of binding CD22.

[0060] As described herein, the density of binding moieties attached to the polymer can be varied. Therefore, in some embodiments, the binding moiety is attached to the PIC polymer at a density of at least one binding moiety about each 400 nm. In some embodiments, the binding moiety is attached to the PIC polymer at a density of at least one binding moiety about each 200 nm - that is, the conjugates comprises about one binding moiety per 200 nm, so a polymer of about 400 nm would comprise about two binding moieties. In some embodiments, the binding moiety is attached to the PIC polymer at a density in the range of from one binding moiety about each 400nm to one binding moiety about each 20 nm. In some embodiments the binding moiety is attached to the PIC polymer at a density in the range of from one binding moiety about each 190 nm to one binding moiety about each 40 nm. In some embodiments, the binding moieties are attached at a density of one binding moiety about each 190 nm to about each 40 nm, preferably about each 130 nm to about each 90 nm. In one embodiment, the binding moieties are attached at a density of one binding moiety about each 40 nm. In one embodiment, the binding moieties are attached at a density of one binding moiety about each 90 nm. In one embodiment, the binding moieties are attached at a density of one binding moiety about each 130 nm. In one embodiment, the binding moieties areattached at a density of one binding moiety about each 190 nm.

[0061] The density of binding moieties can be varied by modifying the quantity of binding moieties present in this reaction relative to the quantity of functional moieties. In such embodiments where attachment of the binding moieties is via the functional moieties of the polymer, density of a binding moiety can be varied by changing the amount of the binding moiety relative to the PIC polymer bearing the functional moiety. In some embodiments, the density of the binding moiety can be varied by varying the ratio of functionalised to non-functionalised monomers during polymerisation, thereby varying the subsequent density of attachment of binding moieties. In some of such embodiments, this density can be further modified by the frequency of conversion of functionalised monomers, for example conversion to moieties such as biotin or streptavidin.

[0062] It will be appreciated by the skilled person that polymerisation is a random process, as is the attachment of binding moieties to the PIC polymer. Therefore, when conjugates are produced, the above-described densities and number of binding moieties refer to the mean density or number of binding moieties.

[0063] Composition

[0064] Also provided is a composition comprising the conjugate of the invention, further comprising a pharmaceutically acceptable excipient, for example an aqueous carrier such as water or saline. Preferably the conjugate is comprised in a composition, for example a composition suitable for administration of the conjugate. Preferably, a composition comprises of a conjugate as described herein and one or more suitable pharmaceutically acceptable excipients. Compositions useful in the methods of the present disclosure include those suitable for various routes of administration, including, but not limited to, intravenous, subcutaneous, intradermal, subdermal, intranodal, intratumoral, intramuscular, intraperitoneal, oral, nasal, topical (including buccal and sublingual), rectal, vaginal, aerosol and / or parenteral or mucosal application.

[0065] The term "pharmaceutically acceptable excipient", as used herein, is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration (see e.g. “Handbook of Pharmaceutical Excipients”, Rowe et al eds. 9th edition, 2020, www.pharmaceuticalpress.com). The use of such media and agents for pharmaceutically active substances is well known in the art. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-forming counter ions such as sodium; metal complexes (e.g. Zn2+-protein complexes); and / or non-ionic surfactants such as polysorbate, block copolymers of poly(alkylene glycols) such as poloxamers, or polyethylene glycol (PEG).

[0066] Cancer

[0067] A cancer can be haematological cancer, neuroblastoma, glioma, lung cancer, bladder cancer, renal cancer, pancreatic cancer, adenocarcinoma, or epithelial cancer, and more preferably haematological cancer. Preferred examples of epithelial cancer are colorectal cancer, breast cancer, head and neck cancer, and prostate cancer.

[0068] In some embodiments, the cancer is a haematological cancer. A haematological cancer is a cancer that originates in blood-forming tissue, such as the bone marrow or cells of the immune system. In some embodiments, the haematological cancer is leukemia, myeloma or lymphoma. The term “leukemia” includes acute lymphoblastic leukmia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML) and acute monocytic leukemia (AMoL). When the cancer is leukemia, the leukemia is preferably chronic lymphocytic leukemia. The term “myeloma” includes multiple myeloma, light chain myeloma or Bence-Jones myeloma, non secretory myeloma, smouldering myeloma, solitary plasmacytoma, extramedullary plasmacytoma, and various myeloma subtypes such as IgG kappa, IgA kappa, IgD kappa, IgE kappa, IgM kappa, IgG lambda, IgA lambda, IgD lambda, IgE lambda and IgM lambda. When the cancer is myeloma, the myeloma is preferably multiple myeloma. The term “lymphoma” includes Hodgkin lymphoma, Epstein-Barr virus-associated lymphoproliferative disease and non-Hodgkin lymphoma. In some embodiments wherein the cancer is lymphoma, the lymphoma is preferably non-Hodgkin lymphoma, more preferably B-cell non-Hodgkin lymphoma, most preferably diffuse large B cell lymphoma.

[0069] In some embodiments, the haematological cancer is a lymphoma, more preferably a non-Hodgkin lymphoma, such as diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, marginal zone B-cell lymphoma (MZL) or mucosa-associated lymphatic tissue lymphoma (MALT), small lymphocytic lymphoma (SLL) and mantle cell lymphoma (MCL). In some embodiments, the haematological cancer is diffuse large B-cell lymphoma (DLBCL) or variants or sub-types thereof, such as primary mediastinal B-cell lymphoma, T cell / histiocyte-rich large B-cell lymphoma, primary cutaneous diffuse large B-cell lymphoma leg type, Epstein-Barr virus positive diffuse large B-cell lymphoma of the elderly, diffuse large B-cell lymphoma associated with chronic inflammation, fibrin-associated diffuse large B-cell lymphoma, primary testicular diffuse large B-cell lymphoma. In some embodiments, the lymphoma is a B-cell lymphoma, such as diffuse large B-cell lymphoma (DLBCL); follicular lymphoma; marginal zone B-cell lymphoma (MZL) or mucosa-associated lymphatic tissue lymphoma (MALT); small lymphocytic lymphoma (SLL); mantle cell lymphoma (MCL); DLBCL variants or sub-types of such as primary mediastinal B-cell lymphoma, T cell / histiocyte-rich large B-cell lymphoma, primary cutaneous diffuse large B-cell lymphoma leg type, Epstein-Barr virus positive diffuse large B-cell lymphoma of the elderly, diffuse large B-cell lymphoma associated with chronic inflammation, fibrin-associated diffuse large B-cell lymphoma, primary testicular diffuselarge B-cell lymphoma; Burkitt lymphoma; lymphoplasmacytic lymphoma, which may manifest as Waldenstrom's macroglobulinemia; nodal marginal zone B cell lymphoma (NMZL); splenic marginal zone lymphoma (SMZL); intravascular lymphomas variants such as intravascular large B-cell lymphoma, intravascular NK-cell lymphoma and intravascular T-cell lymphoma; primary effusion lymphoma; lymphomatoid granulomatosis; primary central nervous system lymphoma; ALK+ large B-cell lymphoma; plasmablastic lymphoma; large B-cell lymphoma arising in HHV8-associated multicentric Castleman's disease; unclassifiable B-cell lymphoma with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma, or with features intermediate between diffuse large B-cell lymphoma and classical Hodgkin lymphoma.

[0070] In some embodiments, the haematological cancer is a lymphoma, a leukemia, or a myeloma, preferably a lymphoma or a leukemia, more preferably a lymphoma, wherein the haematological cancer expresses or is capable of expressing CD20. In preferred embodiments, the haematological cancer expressing or capable of expressing CD20 is a B-cell lymphoma or a B-cell leukemia, preferably a B-cell lymphoma, wherein the B-cell lymphoma is preferably a B-cell non-Hodgkin lymphoma, and wherein the B-cell non-Hodgkin lymphoma is preferably diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone lymphoma, Burkitt lymphoma, (small) lymphocytic lymphoma, (chronic) lymphocytic leukemia, hairy cell leukemia, or primary mediastinal B-cell lymphoma, more preferably diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone lymphoma, (small) lymphocytic lymphoma, most preferably it is diffuse large B-cell lymphoma.

[0071] In some embodiments, the haematological cancer is a lymphoma, a leukemia, or a myeloma, preferably a lymphoma or a leukemia, more preferably a lymphoma, wherein the haematological cancer expresses or is capable of expressing CD37. In preferred embodiments, the haematological cancer expressing or capable of expressing CD37 is a B-cell lymphoma or a B-cell leukemia, preferably a B-cell lymphoma, wherein the B-cell lymphoma is preferably a B-cell non-Hodgkin lymphoma, and wherein the B-cell non-Hodgkin lymphoma is preferably diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone lymphoma, Burkitt lymphoma, (small) lymphocytic lymphoma, (chronic) lymphocytic leukemia, hairy cell leukemia, or primary mediastinal B-cell lymphoma, more preferably diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone lymphoma, (small) lymphocytic lymphoma, most preferably it is diffuse large B-cell lymphoma.

[0072] In some embodiments, the haematological cancer is a lymphoma, a leukemia, or a myeloma, preferably a lymphoma or a leukemia, more preferably a lymphoma, wherein the haematological cancer expresses or is capable of expressing CD20 and CD37. In preferred embodiments, the haematological cancer expressing or capable of expressing CD20 and CD37 is a B-cell lymphoma or a B-cell leukemia, preferably a B-cell lymphoma, wherein the B-cell lymphoma is preferably a B-cell non-Hodgkin lymphoma, and wherein the B-cell non-Hodgkin lymphoma is preferably diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone lymphoma, Burkitt lymphoma, (small) lymphocytic lymphoma, (chronic) lymphocytic leukemia, hairycell leukemia, or primary mediastinal B-cell lymphoma, more preferably diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone lymphoma, (small) lymphocytic lymphoma, most preferably it is diffuse large B-cell lymphoma.

[0073] In some preferred embodiments the cancer, preferably a haematological cancer, is preferably a lymphoma, a leukemia, or a myeloma, wherein the cancer expresses or is capable of expressing CD20 and CD37. In some preferred embodiments the cancer, preferably a haematological cancer, is preferably a lymphoma, a leukemia, or a myeloma, wherein the cancer expresses oris capable of expressing CD20 and CD22. In some preferred embodiments the cancer, preferably a haematological cancer, is preferably a lymphoma, a leukemia, or a myeloma, wherein the cancer expresses or is capable of expressing CD22 and CD37.

[0074] In some embodiments, the haematological cancer is a myeloma, a leukemia, or a lymphoma, preferably a myeloma or a lymphoma, more preferably a myeloma, wherein the haematological cancer expresses or is capable of expressing CD38. Examples of haematological cancers expressing or capable of expressing CD38 are plasma cell cancers, such as (active) multiple myeloma, plasma cell leukemia, plasmablastic lymphoma, solitary plasmacytoma, and extramedullary plasmacytoma; B-cell leukemia and / or B-cell lymphoma, such as (chronic) lymphocytic leukemia, (small) lymphocytic lymphoma, hairy cell leukemia, Burkitt lymphoma, diffuse large B-cell lymphoma, and primary mediastinal B-cell lymphoma; T-cell leukemia and / or T-cell lymphoma, such as T-cell acute lymphoblastic leukemia (T-ALL), T-cell lymphoblastic lymphoma (T-LBL), and peripheral T-cell lymphoma (PTCL); and myeloid malignancies, such as acute myeloid leukemia (AML), and acute promyelocytic leukemia (APL). In preferred embodiments, the haematological cancer expressing or capable of expressing CD38 is a plasma cell cancer, wherein the plasma cell cancer is multiple myeloma, plasma cell leukemia, plasmablastic lymphoma, solitary plasmacytoma, or extramedullary plasmacytoma, preferably multiple myeloma, plasma cell leukemia, or plasmablastic lymphoma, more preferably multiple myeloma, or plasma cell leukemia, most preferably it is multiple myeloma. The haematological cancer expressing or capable of expressing CD38 may be symptomatic or active, as well as asymptomatic. Examples of asymptomatic haematological cancers expressing or capable of expressing CD38 are smoldering myeloma and monoclonal gammopathy of undetermined significance (MGUS).

[0075] In preferred embodiments, the haematological cancer expressing or capable of expressing CD38 is multiple myeloma. In some embodiments, the multiple myeloma is IgG myeloma, IgA myeloma, IgD myeloma, IgE myeloma, IgM myeloma, light chain myeloma, Bence-Jones myeloma, or non-secretory myeloma, preferably IgG myeloma, IgA myeloma, light chain myeloma, or Bence-Jones myeloma, more preferably IgG myeloma.

[0076] The multiple myeloma may be symptomatic or asymptomatic, preferably it is symptomatic. Preferably, the multiple myeloma is symptomatic multiple myeloma, wherein the symptomatic multiple myeloma is defined by the presence of one or more CRAB criteria. The CRAB criteria refer to clinical indicators of organ damage caused by cancer cells, and specifically refer to hypercalcemia, renal failure, anemia, or bone lesions. Accordingly, in some embodiments, thesymptomatic multiple myeloma is associated with hypercalcemia, wherein the hypercalcemia is preferably defined by a serum calcium concentration of above 2.75 mmol / L blood, and / or a serum calcium concentration of more than 0.25 mmol / L blood above the upper limit of the normal serum calcium range in the subject, as measured in a venous blood sample of a subject. In some embodiments, the symptomatic multiple myeloma is associated with renal impairment, wherein the renal impairment is preferably defined by a serum creatinine concentration of above 177 pmol / L blood, and / or a serum creatinine concentration of more than 40% above the upper limit of the normal serum creatinine range in a subject, as measured in a venous blood sample of a subject. In some embodiments, the symptomatic multiple myeloma is associated with anemia, wherein anemia is preferably defined by a haemoglobin value of below 100 g / L blood, and / or a haemoglobin value of at least 20 g / L blood below the lower limit of the normal haemoglobin value of a subject, as measured from a complete blood count of a venous blood sample of a subject. In some embodiments, the symptomatic multiple myeloma is associated with one or more bone lesions, wherein the one or more bone lesions are preferably defined as one or more sites of osteolytic bone destruction of at least 5 mm in size as identified by radiological imaging, preferably by CT or18F-fluorodeoxyglucose PET with CT (PET-CT).

[0077] In some embodiments, the multiple myeloma is hyperdiploid multiple myeloma. Hyperdiploid multiple myeloma is characterized by the presence of one or more chromosomal trisomies, such as trisomy of chromosomes 3, 5, 7, 9, 11 , 15, 19, and / or 21. Preferably, the hyperdiploid multiple myeloma comprises at least 2, preferably at least 3, more preferably at least 4 trisomies of chromosomes 3, 5, 7, 9, 11 , 15, 19, and / or 21. In some embodiments, the multiple myeloma is non-hyperdiploid multiple myeloma. Non-hyperdiploid multiple myeloma is characterized by chromosomal translocations, referred to as “t”, involving the immunoglobulin heavy chain (IgH) locus on chromosome 14, such as t(11 ;14), t(4;14), t(14;16), and t(14;20), preferably t(11 ; 14) and t(4; 14), more preferably t(11 ; 14) . In some embodiments, the non-hyperdiploid multiple myeloma is characterized by one or more of the following translocations: t(11 ;14)(q13;q32), t(4;14)(p16;q32), t(14; 16)(q32;q23), and t(14;20)(q32;q11). As used herein, “t(a;b)(x;y)” refers to a translocation (“t”) between chromosome a and chromosome b, wherein the breakpoints occur at specific bands x and y on the respective chromosomes. For example, t(11 ;14)(q13;q32) indicates a translocation between chromosome 11 at band q13, and chromosome 14 at band q32.

[0078] The conjugate of the invention is capable of binding a membrane protein on a cancer cell. As used herein, “a cancer cell” represents a cell that originates from a cancer, and exemplary cancers that are relevant in the context of the present invention are set out above. In some embodiments, the conjugate is capable of binding a haematological cancer cell, preferably a lymphoma cell, a chronic lymphocytic leukemia cell, or a multiple myeloma cell, more preferably a non-Hodgkin lymphoma cell.

[0079] In some embodiments, the cancer cell is characterized by reduced, preferably the absence of, apoptotic markers, such as expression and / or activation of p38, Bax (Bcl-2-associated X protein), Bim (Bcl-2-interacting mediator of cell death), p53, Fas / FasL, Caspase-3, Caspase-8, Caspase-9, GADD45, Calpain, Caspase-12, CHOP (C / EBP Homologous Protein) and Bad (Bcl-2-associated Death Promoter). In some embodiments, the cancer cell is characterized by reduced levels of phosphorylated and / or activated p38 as compared to a non-cancer cell or a healthy cell of the same origin, preferably as measured by phosphor flow cytometry. As used herein, ”p38” refers to p38 mitogen-activated protein kinase, in particular to p38 mitogen-actived protein kinase alpha (p38a). Accordingly, in some embodiments, the conjugates of the invention are for promoting p38 phosphorylation in the cancer cell. For example, p38 phosphorylation in the cancer cell is increased by a factor between 2 and 100, more preferably between 10 and 80, more preferably between 20 and 60, such as about 30 to 50, or 35 to 40, relative to the extent of p38 phosphorylation observed when a control antibody, preferably an isotype control antibody, is applied. Preferably, the control antibody is present in soluble form, i.e. not immobilized on for example a PIC polymer. In some embodiments, the conjugates of the invention promote p38 phosphorylation in the cancer cell up to about 2 to 20%, preferably 5 to 15%, more preferably about 8 to 12%, such as 10%, of the total p38 levels. Levels are preferably determined as demonstrated in the Examples.

[0080] In some embodiments, the cancer cell is characterized by reduced levels of intracellular calcium (Ca2+) as compared to a non-cancer cell or healthy cell of the same origin, preferably as measured by phosphor flow cytometry. In some embodiments, the conjugates of the invention are for promoting intracellular Ca2+levels in a cancer cell. Preferably, the conjugates of the invention promote Ca2+levels in a cancer cell about 1.2 to 2.5 times, more preferably about 1.3 to 2 times, most preferably about 1.5 times, in comparison to a control antibody, preferably an isotype control antibody.

[0081] In some embodiments, the cancer cell is characterized by reduced levels of cleaved caspase-3 as compared to a non-cancer cell or healthy cell of the same origin, preferably as measured by phosphor flow cytometry, more preferably as described in the Examples. In some embodiments, the conjugates of the invention are for promoting cleaved caspase-3 levels in a cancer cell. Preferably, the conjugates of the invention increase cleaved caspase-3 levels in a cancer cell about 1.2 to 3 times, more preferably about 1.3 to 2.5 times, even more preferably about 1.4 to 2.2 times. Preferably the conjugates of the invention increase cleaved caspase-3 levels in a cancer cell at least about 1.5 times, preferably at least about 1.7 times, most preferably at least about 2 times.

[0082] Therapeutic use

[0083] The invention provides a conjugate as described herein for use as a medicament. In some embodiments, the medicament is for treating cancer, preferably haematological cancer such as lymphoma, chronic lymphocytic leukemia, or multiple myeloma, even more preferably non-Hodgkin lymphoma, most preferably diffuse large B cell lymphoma. In one embodiment, the conjugate is used as an active ingredient, component or substance in a medicament. Preferably, the conjugates are for use as a medicament, wherein the medicament is for treating cancer. Accordingly, the conjugates can be used in an anti-cancer therapy, preferably of cancers as described earlier herein.

[0084] In a further aspect there is provided a method for treating cancer in a subject, the method comprising the step of administering the conjugate according to the invention to the subject.Preferably, the subject is a human subject. In some embodiments, the conjugate is administered in the form of a pharmaceutical preparation comprising the conjugate as an active ingredient. In some embodiments, the treatment can comprise the steps of a) identifying a membrane protein that is expressed by (tumor) cells in the cancer; b) selection of a conjugates as described herein that specifically bind the membrane protein that is expressed by (tumor) cells in the cancer; c) using the selected conjugate in the treatment of the cancer. The cancer can be a cancer as described above.

[0085] In some embodiments, the conjugate according to the invention can be used in combination therapies with one or more other therapeutic agents. Such therapeutic agents when used in the treatment of cancer, include, but are not limited to anti-cancer agents and chemotherapeutic agents. Exemplary therapeutic agents that may be used as part of a combination therapy in treating cancer, include, for example, radiation, mitomycin, tretinoin, ribomustin, gemcitabine, vincristine, etoposide, cladribine, mitobronitol, methotrexate, doxorubicin, carboquone, pentostatin, nitracrine, zinostatin, cetrorelix, letrozole, raltitrexed, daunorubicin, fadrozole, fotemustine, thymalfasin, sobuzoxane, nedaplatin, cytarabine, bicalutamide, vinorelbine, vesnarinone, aminoglutethimide, amsacrine, proglumide, elliptinium acetate, ketanserin, doxifluridine, etretinate, isotretinoin, streptozocin, nimustine, vindesine flutamide, drogenil, butocin, carmofur, razoxane, sizofilan, carboplatin, mitolactol, tegafur, ifosfamide, prednimustine, picibanil, levamisole, teniposide, improsulfan, enocitabine, lisuride, oxymethoIone, tamoxifen, progesterone, mepitiostane, epitiostanol, formestane, interferon-alpha, interferon-2 alpha, interferon-beta, interferon-gamma, colony stimulating factor-1 , colony stimulating factor-2, denileukin diftitox, interleukin-2, and luteinizing hormone releasing factor.

[0086] An additional class of agents that may be used as part of a combination therapy in treating cancer is immune checkpoint inhibitors. Exemplary immune checkpoint inhibitors include agents that inhibit one or more of (i) cytotoxic T-lymphocyte-associated antigen 4 (CTLA4), (ii) programmed cell death protein 1 (PD1), (iii) PD-L1 , (iv) LAG3, (v) B7-H3, (vi) B7-H4, and (vii) TIM3. Yet other agents that may be used as part of a combination therapy in treating cancer are monoclonal antibodies against membrane proteins as described herein. In some embodiments the administration of the conjugates of the invention and the other therapeutic agent can elicit an additive or synergistic effect on immunity and / or on therapeutic efficacy.

[0087] The administration route of the conjugates as described herein can be parenteral. This includes intravenous, intra-arterial, intralymphatic, intraperitoneal, intramuscular or subcutaneous. The intravenous or intramuscular forms of parenteral administration are preferred. The amount of conjugate required for therapeutic or prophylactic effect will, of course, vary with the conjugate chosen, the nature and severity of the condition being treated and the patient. Preferably the dosing is given by injections, most preferably intravenous, intramuscular or subcutaneous injections, depending in part on whether the administration is brief or chronic. Preferably an effective dose is used.

[0088] General definitions

[0089] Various terms relating to the methods, compositions, uses and other aspects of the presentinvention are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art to which the invention pertains, unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definition provided herein. Although any methods and materials similar or equivalent to those described herein can be used in the practice fortesting of the present invention, the preferred materials and methods are described herein. All cited publications are incorporated herein by reference.

[0090] “A,” “an,” and “the”: these singular form terms include plural referents unless the content clearly dictates otherwise. The indefinite article “a” or “an” thus usually means “at least one”. Thus, for example, reference to “a cell” includes a combination of two or more cells, and the like.

[0091] “About” and “approximately”: these terms, when referring to a measurable value such as an amount, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods. Additionally, numerical values are sometimes presented herein in a range format. It is to be understood that such range format is used for convenience and brevity and should be understood flexibly to include numerical values explicitly specified as limits of a range, but also to include all individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly specified.

[0092] “And / or”: The term “and / or” refers to a situation wherein one or more of the stated cases may occur, alone or in combination with at least one of the stated cases, up to with all of the stated cases. “Comprising”: this term is construed as being inclusive and open ended, and not exclusive. Specifically, the term and variations thereof mean the specified features, steps or components are included. These terms are not to be interpreted to exclude the presence of other features, steps or components. “Exemplary”: this term means “serving as an example, instance, or illustration,” and should not be construed as excluding other configurations disclosed herein.Description of the figures

[0093] iq. 1A SDS-PAGE gel demonstrating purity of IFs. Each slot was loaded with 0.37 pg IF or soluble antibody in non-reducing sample buffer. Left gel represents aCD20 signal; right gel represents aCD37 signal. Purity of the IFs, indicated by the number below the lanes, was calculated by dividing the signal around 150 kDa (aCD20) or 100 kDa (aCD37), i.e., unconjugated soluble antibody present in the sample, by the total lane signal.

[0094] iq. 1B Binding of isotype control antibody, soluble aCD20 and aCD37, and aCD20-, aCD37-, and aCD20 / 37 IFs to OCI-LY1 cells after 30-minute incubation at 4 °C. Shown are representative flow cytometry histograms of bound aCD20 (left, AF647 signal) and aCD37 (right, AF488 signal).

[0095] The geometric mean fluorescence intensity (gMFI) of the IFs was divided by the gMFI of the corresponding soluble antibody to calculate the percentage of bound IF compared to soluble antibody, as shown in the bar graph. Shown are the mean ± SEM and individual data points of three independent experiments.

[0096] Representative Airyscan confocal images of OCI-LY1 cells after treatment with aCD20 and aCD37 (top) or aCD20 / 37-IF (bottom) for 30 minutes at 37 °C. Displayed are nuclei (far left), CD20 (mid left), CD37 (mid right), and a merged image (far right). Scalebar: 5 pm.

[0097] iq. 2A CDC of OCI-LY1 cells after 3-hour incubation with filtered aCD20- or aCD37-IFs, unfiltered aCD20 / 37-IFs, or corresponding soluble antibodies in RPMI1640 media supplemented with 1% stable glutamine and 40% heat inactivated pooled human serum or 40% active pooled human serum at 37 °C. Cell death was determined by cell viability staining (Zombie Violet) using flow cytometry, corrected for cell death induced by an isotype control antibody in heat-inactivated pooled human serum or active pooled human serum. Shown are the mean ± SEM and individual data points of two independent experiments. Statistical significance was determined using a two way ANOVA with Bonferroni post-hoc test. *p<0.05, **p<0.01.

[0098] iq. 2B Direct cell death of OCI-LY1 cells after 24-hour incubation with filtered aCD20-, aCD37-, or aCD20 / 37-IFs, corresponding soluble antibodies, obinutuzumab (OBZ) or blank IF (bIF) in RPMI1640 media supplemented with 1% stable glutamine and 10% heat-inactivated fetal bovine serum at 37 °C. Cell death was determined by cell viability staining (Zombie Violet) using flow cytometry, corrected for cell death induced by an isotype control antibody. Shown are the mean ± SEM and individual data points of three independent experiments. Statistical significance was determined using a one-way ANOVA with Tukey’s post-hoc test. *p<0.05, **p<0.01 , ***p< 0.0001. Abbreviations: ns, non-significant.

[0099] iq. 3A SDS-PAGE gel demonstrating 97% purity of batch B aCD20-IF (aCD20-IF-B). Slots were loaded with 0.36 pg soluble aCD20 oraCD20-IF-B in non-reducing sample buffer. Purity of aCD20- IF-B was calculated by dividing the signal around 150 kDa (aCD20), displaying unconjugated soluble aCD20, by the total lane signal.

[0100] iq. 3B Binding of aCD20-IF-B to OCI-LY1 cells after 30-minute incubation at 4 °C. The geometric mean fluorescence intensity (gMFI) of aCD20-IF-B was divided by the gMFI of soluble aCD20 tocalculate the percentage of bound IF compared to soluble aCD20. Shown are the mean ± SEM and individual data points of four independent experiments.

[0101] iq. 3C Representative Airyscan confocal images of OCI-LY1 cells after treatment with aCD20 (top) or aCD20-IF-B (bottom) for 30 minutes at 37 °C. Displayed are nuclei (left) and CD20 (right). Scalebar: 5 pm.

[0102] Fig. 4A - Direct cell death of OCI-LY1 cells after 24 hour incubation with soluble aCD20 (corresponding to aCD20-IF-B), aCD20-IF-A, or aCD20-IF-B in RPMI1640 media supplemented with 1% stable glutamine and 10% heat-inactivated fetal bovine serum at 37 °C. Cell death was determined by cell viability staining (Zombie Violet) using flow cytometry, corrected for cell death induced by an isotype control antibody. Shown are the mean ± SEM and individual data points of five independent experiments. Statistical significance was determined using a one-way ANOVA with Tukey’s post-hoc test. **p<0.01 , ***p<0.001. Abbreviations: ns, non-significant.

[0103] iq. 4B Percentage of apoptotic cells in OCI-LY1 cells after 24 hour incubation with soluble aCD20 (corresponding to aCD20-IF-B), aCD20-IF-A, or aCD20-IF-B in RPMI1640 media supplemented with 1% stable glutamine and 10% heat-inactivated fetal bovine serum at 37 °C. The percentage of apoptotic cells was determined via Annexin V-PE-Cy7 staining using flow cytometry, corrected for cell death induced by an isotype control antibody. Shown are the mean ± SEM and individual data points of four independent experiments. Statistical significance was determined using a one-way ANOVA with Tukey’s post-hoc test. *p<0.05. Abbreviations: ns, non-significant.

[0104] iq. 5A Expression of phosphorylated p38 (p-p38) in OCI-LY1 cells after 1-hour pre incubation with 0.0001% DMSO (vehicle) or SB203850, a p38 inhibitor, followed by 4-hour incubation in RPMI1640 media supplemented with 1% stable glutamine and 10% heat-inactivated fetal bovine serum containing soluble aCD20 or aCD20-IF-B at 37 °C. Cells were stained with primary antibody rabbit anti-p-p38-MAPK and with secondary antibody phycoerythrin(PE)-conjugated donkey antirabbit. The bar graphs show the mean ± SEM and individual data points of the percentage of p-p38(P Expositive cells from three independent experiments determined by phospho flow cytometry, corrected for p-p38 expression induced by an isotype control antibody. Statistical significance was determined using a two-way ANOVA with Tukey’s post-hoc test. *p<0.05, **p<0.01.

[0105] iq. 5B Induction of direct cell death in OCI-LY1 cells after 1-hour pre incubation with 0.0001% DMSO (vehicle) or SB203850, a p38 inhibitor, followed by 4-hour incubation in RPMI1640 media supplemented with 1% stable glutamine and 10% heat-inactivated fetal bovine serum containing soluble aCD20 or aCD20-IF-B at 37 °C. Cells were stained with a viability dye (Zombie Violet) and subsequently fixed. The bar graphs show the mean ± SEM and individual data points of the percentage cell death from three independent experiments determined by phospho flow cytometry, corrected cell death induced by an isotype control antibody. Statistical significance was determined using a two-way ANOVA with Tukey’s post-hoc test. **p<0.01 , ****p<0.0001.

[0106] iq. 5C Fluo-4 expression in OCI-LY1 cells after 4-hour incubation in RPMI1640 media supplemented with 1% stable glutamine and 10% heat-inactivated fetal bovine serum containing an isotype control antibody, soluble aCD20, oraCD20-IF-B at 37 °C, followed by 30-minute incubationin Fluo-4-AM, an intracellular Ca2+indicator, at 37 °C. Shown are representative flow cytometry histograms of expression of Fluo-4.

[0107] Fig. 5D - Quantification of the experiment according to Fig. 5C. The bar graph shows the mean ± SEM and individual data points of the geometric mean fluorescence intensity (gMFI) ratio over isotype control antibody from three independent experiments. Statistical significance was determined using an unpaired t-test. **p<0.01.

[0108] Fig. 6A-The IF features a polyisocyanopeptide (PIC) backbone (grey curved line) of approximately 400 nm in length. Therapeutic antibodies are modified with AZDye 647 (AZ647) dyes and a dibenzocyclooctyne (DBCO) group by which the antibody is bound to the PIC via bioorthogonal click chemistry through reactive azide monomers (N3) present in the side chains.

[0109] Fig. 6B - Flow cytometry histogram showing daratumumab-IFs and isatuximab-IFs or their corresponding soluble antibodies (all at 5 pg / ml) binding to CD38 in the RPMI-8226 cell line. From top to bottom, the histogram corresponds to: unstained cells, daratumumab soluble, daratumumab-IF, isatuximab soluble, and isatuximab-IF. The quantification of daratumumab-IFs, and their corresponding soluble antibody is shown in the middle panel, and the quantification of isatuximab-IFs and their corresponding soluble antibody is shown in the right panel (geometric mean fluorescence intensity, gMFI). All datapoints are derived from 3 independent experiments and graphs depict mean values ± SD. Statistical significance was assessed by paired t-test (*p<0.05, **p<0.01 , ns=non-significant).

[0110] Fig. 6C - As in Fig. 6B, using the LP-1 cell line.

[0111] Fig. 6D - As in Fig. 6B, using the DAUDI cell line.

[0112] Fig. 6E - As in Fig. 6B, using primary MM cells.

[0113] Fig. 6F - Histogram as in Fig. 6B, using the CD38-negative KM-H2 cell line, as a control for specific binding.

[0114] Fig. 6G - Internalization was quantified by flow cytometry, as the percentage of surface-bound daratumumab-IFs or isatuximab-IFs (or their soluble counterparts) remaining after acid stripping following incubation at 37 °C for the indicated times in RPMI-8226 cells, based on gMFI values normalized to 4 °C controls. All datapoints are derived from 3 independent experiments and graphs depict mean values ± SD. Statistical significance was assessed by repeated measures one-way ANOVA with Tukey’s multiple comparisons test (*p<0.05, **p<0.01 , ns=non-significant).

[0115] Fig. 6H - As in Fig. 6G, but using the LP-1 cell line.

[0116] Fig. 6I - As in Fig. 6G, but using the DAUDI cell line.

[0117] Fig. 7A - LP-1 cells were treated with daratumumab (dara)-IFs or isatuximab (isa)-IFs or their corresponding soluble antibodies (all at 5 pg / ml), and cell death was analyzed by flow cytometry. Graphs plot the quantification of dead cells (propidium iodide+) after treatment with dara-IFs (left panel) or isa-IFs (right panel) for 3 h at 37 °C in the presence of human serum (dark grey bars, individual data points shown by squares) or its heat-inactivated form (light grey bars, individual data points shown as circles) in LP-1 cells. Soluble dara antibodies (left panel) or isa antibodies (right panel), isotype control, or no treatment (NT) served as control. Data derived from 3 independent experiments. The graphs depict mean values ± SD. Statistical significance was assessed byrepeated measures two-way ANOVA with Tukey’s.

[0118] Fiq. 7B - As in Fig. 7A, but using DAUDI cells instead of LP-1 cells. The data relating to Isa-treated DAUDI cells, i.e. in the right panel, is derived from 4 independent experiments.

[0119] Fiq. 7C - As in Fig. 7A, i.e. in LP-1 cells, but using control IFs without any antibodies.

[0120] Fiq. 7D - As in Fig. 7B, i.e. in DAUDI cells, but using control IFs without any antibodies.

[0121] Fiq. 8A - CD38 cluster analysis by quantification of the number of CD38 clusters per cell area Airyscan confocal images after treatment with daratumumab-IFs or soluble antibodies (all at 5 pg / ml) for 30 min at 37 °C. Datapoints are 64 and 61 cells for soluble daratumumab and daratumumab-IFs, respectively, derived from 3 independent experiments. Statistical significance was assessed by unpaired t-test with Welch’s correlation for cluster number.

[0122] Fiq. 8B - Quantification of the size of the CD38 clusters counted in Fig. 8A. Statistical significance was assessed by Mann Whitney U-test for cluster size. Mean + / - SD is shown.

[0123] Fiq. 8C - As in Fig. 8A, but after treatment with isatuximab-IFs or soluble antibodies. Datapoints are 68 and 69 cells for soluble isatuximab and isatuximab-IFs, respectively, derived from 3 independent experiments.

[0124] Fiq. 8D - As in Fig. 8B, but after treatment with iratuximab-IFs or soluble antibodies.

[0125] Fiq. 9 - Cell death induced by daratumumab-IF in the presence of NK cells. RPMI-8226 cells were treated with soluble daratumumab (dara), dara-IF, isotype control (all at 5 pg / ml), or no treatment (NT), for 16 h at 37 °C in the presence or absence of NK cells (1 :1 ratio). Quantification of dead cells (eF780 +) was performed by flow cytometry. Data are from 3 NK cell donors and 2 independent experiments. Bars represent mean ± SD. Statistical significance was assessed by repeated measures one-way ANOVA between conditions where NK cells were present. (**p<0.01 , ****p<0.0001).

[0126] Fiq. 10A - Flow cytometry gating strategy for apoptotic cell identification in RPMI-8226 cells, showing data of the cells treated with isatuximab (isa)-IF or soluble isa. Total population was selected first (upper left panel), followed by single-cell selection (upper right panel), and by representative flow plots after 24 h treatment (lower panels).

[0127] Fiq. 10B - RPMI-8226 cells were treated with dara-IFs or their corresponding soluble antibodies, or control IF (no antibody conjugation) for 6 or 24 h at 37 °C, and stained with Annexin V-PE and eF450. Quantification of early apoptotic (Annexin V-PE +) and late apoptotic / necrotic (Annexin V-PE + / eF450 +) RPMI-8226 cells was performed according to the gating strategy in Fig. 10A. Data values were normalized by subtracting isotype control values. All graphs depict mean values ± SD. Statistical significance was assessed by mixed effects analysis with Tukey’s multiple comparisons test (*p<0.05, **p<0.01 , ***p<0.001 , ****p<0.0001).

[0128] Fiq. 10C - As in Fig. 10B, in LP-1 cells.

[0129] Fiq. 10D - As in Fig. 10B, in DAUDI cells.

[0130] Fiq. 10E - As in Fig. 10B, with isa-IFs and their corresponding soluble antibodies instead of dara-IFs and their corresponding antibodies.

[0131] Fig. 10F - As in Fig. 10E, in LP-1 cells.

[0132] Fig. 10G - As in Fig. 10E, in DAUDI cells.

[0133] Fig. 11A - RPMI-8226 cells were treated with dara-IFs or isa-IFs or their corresponding soluble antibodies for 6 h at 37 °C and stained for cleaved caspase-3, a marker of apoptosis. The bar graph represents the quantification ofcleaved caspase-3 in RPMI-8226 cells after treatment with dara-IFs or isa-IFs compared to their soluble forms. Data derived from 4 independent experiments. Graphs depict mean values ± SD. Statistical significance was assessed by Sidak’s multiple comparisons test (*p<0.05, **p<0.01).

[0134] Fig. 11B - LP-1 cells were treated with dara-IFs or isa-IFs or their corresponding soluble antibodies (all at 5 pg / ml) for 4 h at 37 °C, and intracellular calcium levels were measured by flow cytometry using Fluo-4-AM and normalized to isotype control-treated cells. The bar graph represents the quantification of intracellular calcium levels from 3 independent experiments. Graphs depict mean values ± SD. Statistical significance was assessed by ratio paired t-test (*p<0.05, **p<0.01). gMFI= geometric mean fluorescence intensity.

[0135] Fig. 11 C - As in Fig. 11 B, in RPMI-8226 cells.

[0136] Fig. 11 D - As in Fig. 11 B, in DAUDI cells.

[0137] Fig. 12A- Histograms showing rituximab-IF or soluble rituximab (both at 10 pg / ml) binding to CD20 in OCI-LY1 cells. From top to bottom, the histogram represents: unstained control, rituximab soluble, rituximab-IFs.

[0138] Fig. 12B - As in Fig. 12A, using RAJI cells.

[0139] Fig. 12C - As in Fig. 12A, using DAUDI cells.

[0140] Fig. 12D - As in Fig. 12A, using CD20-negative OCI-LY19 cells.

[0141] Fig. 12E - Quantification of soluble rituximab (ritux) and ritux-IF (both at 10 pg / ml) binding to OCI-LY1 cells by flow cytometry (geometric mean fluorescence intensity, gMFI). Shown are mean ± SD values from 3 independent experiments. Statistical significance was assessed by paired t-test (*p<0.05).

[0142] Fig. 12F - As in Fig. 12E, using RAJI cells.

[0143] Fig. 12G - As in Fig. 12E, using DAUDI cells.

[0144] Fig. 13A - OCI-LY1 cells were treated with soluble rituximab (ritux), ritux-IF, isotype control (all at 10 pg / ml), control IF or no treatment (NT), in the presence of human serum (dark grey bars, individual data points shown by squares) or its heat-inactivated form (light grey bars, individual data points shown as circles). Cell death was assessed by flow cytometry. The graph shows the quantification of dead (zombie violet +) OCI-LY1 cells. Datapoints derived from 3 independent experiments. Mean + / - SD is shown. Statistical significance was assessed by repeated measures two-way ANOVA with Sidak’s multiple comparisons test (**p<0.01 , ***p<0.001, ****p<0.0001). Fig. 13B - As in Fig. 14A, using RAJI cells.

[0145] Fig. 13C - As in Fig. 14A, using DAUDI cells.

[0146] Fig. 14A - CD20 cluster analysis by quantification of the number of CD20 clusters per cell area from Airyscan confocal images made after treatment with rituximab-IFs or soluble antibodies (all at10 pg / ml) for 30 min at 37 °C. Datapoints are 44 and 36 cells for soluble rituximab and rituximab-IFs, respectively, derived from 2 independent experiments. Statistical significance was assessed by Mann Whitney U-test. Mean + / - SD is shown.

[0147] Fig. 14B - Quantification of the size of the CD20 clusters counted in Fig. 15A. Statistical significance was assessed by Mann Whitney U-test for cluster size. Mean + / - SD is shown.

[0148] Fig. 15A - Rituximab-IF enhances direct tumor cytotoxicity compared to soluble rituximab. Quantification of dead cells (zombie violet +) after 24 h treatment with soluble rituximab, rituximab-IF or control IF (no antibody conjugation) in OCI-LY1 cells. Shown are mean ± SD values from 3 independent experiments. Statistical significance was assessed by ordinary one-way ANOVA with Tukey’s multiple comparisons test (*p<0.05, **p<0.01).

[0149] Fig. 15B - As in Fig. 13A, using RAJI cells.

[0150] Fig. 15C - As in Fig. 13A, using DAUDI cells.

[0151] Fig. 16A- Rituximab-IF increases calcium influx compared to soluble rituximab. OCI-LY1 cells were treated with soluble rituximab or rituximab-IF for 4 h at 37 °C, and intracellular calcium levels were quantified using Fluo-4 AM staining. Shown are mean ± SD values from 3 independent experiments. Statistical significance was assessed by paired t-test (**p<0.01).

[0152] Fig. 16B - Rituximab-IF increases p38 phosphorylation compared to soluble rituximab. OCI-LY1 cells were pre-treated with the p38 inhibitor SB203850 or vehicle (DMSO) for 1 h at 37 °C, followed by treatment with soluble rituximab or rituximab-IF for 4 h at 37 °C. Shown is the mean % of p-p38 + cells ± SD values from 3 independent experiments. Statistical significance was assessed by ordinary two-way ANOVA with Sidak’s multiple comparisons test (**p<0.01 , ***p<0.001 , ****p<0.0001).

[0153] Fig. 16C - As in Fig. 16B, showing the mean % of dead cells (zombie violet +) ± SD values.

[0154] Examples

[0155] Example 1. Abstract

[0156] For over two decades, the standard treatment for diffuse large B-cell lymphoma (DLBCL) has been the combination of rituximab (aCD20) immunotherapy with chemotherapy. Still, high relapse and resistance rates demonstrate the need for novel treatment options that enhance anti-tumor activity. Here, two abundantly expressed membrane proteins in DLBCL, CD20 and CD37, were targeted in a multivalent manner by combining rituximab and otlertuzumab (aCD37) on immunofilaments (IFs) to increase tumor cell cytotoxicity in DLBCL cells. aCD20-IFs, aCD37-IFs, and aCD20 / 37-IFs induced increased direct cell death compared to freely available aCD20 and aCD37, despite lower binding efficiency of IFs to target cells. A trend towards higher direct cell death with aCD20 / 37-IFs compared to aCD20- and aCD37-IFs was observed. Increased levels of phosphorylated p38 and intracellular calcium induced by aCD20-IF hinted at a role for CD20 clustering in aCD20-IF-induced direct cell death. The current study shows the potential of aCD20 / 37-IFs to enhance anti-tumor activity in DLBCL patients.Example 2. Materials and methods

[0157] Cell culture

[0158] The DLBCL cell lines OCI-LY1 (CD20+, CD37+, GCB subtype) and OCI-LY19 (CD20-, CD37-, GCB subtype) were cultured in Roswell Park Memorial Institute (RPMI) 1640 media (Gibco) supplemented with 10% Fetal Bovine Serum (FBS, Cytiva), 2mM stable glutamine (Capricorn scientific), and 1X Antibiotic-Antimycotic (Gibco) in a 37 °C, 5% CO2 incubator. Cells were tested regularly for the presence of mycoplasma and were kept in culture no longer than two months.

[0159] Immunofilament (IF) synthesis

[0160] IFs of batch A were synthesized similarly to previously published protocols (Weiss, L., et al., 2023, doi:10.1021 / acsnano.2c11884). Briefly, isocyanopeptide monomers with an azide- or methoxy end group were polymerized in a 1 :30 ratio, respectively, yielding PICs of -400 nm. Rituximab (anti-CD20, Mabthera, Roche) and otlertuzumab (anti-CD37, Ichorbio) were washed with 50 mM borate buffer (pH 8.5) using Amicon ultra centrifugal filters (30 kDa, Merck), dibenzocyclooctyine (DBCO)-functionalized by addition of 3 equivalents of DBCO-PEG4-NHS ester (Jena BioScience), and labeled with 1.5 equivalents of AZDye™ 647 NHS ester (VectorLabs) or ATTO 488 NHS ester (ATTO-TEC), respectively. Functionalized rituximab (aCD20) and otlertuzumab (aCD37) are further referred to as soluble antibodies for clarity, despite otlertuzumab being an antibody-derived therapeutic protein with slightly different properties compared to conventional monoclonal antibodies. 0.1 or 0.2 equivalents (relative to azide groups) of soluble aCD20 or aCD37 was added to the PICs and rotated for 4-5 hours at room temperature to yield aCD20-, aCD37-, and aCD20 / 37-IFs of different densities (batch A). After purification using tangential flow filtration (TFF), IFs were dissolved in PBS. Conjugated antibody concentrations were determined via fluorescence using a Spark 10M plate reader (Tecan), where soluble antibodies were used as standards. PIC concentrations were determined via circular dichroism spectroscopy (JASCO J-810), after which the average antibody spacing could be calculated while assuming an equal distribution of antibodies over the PIC polymers (Table 1) (Weiss, L., et al., 2023, doi:10.1021 / acsnano.2c11884). For aCD20 / aCD37-IF, the ratio aCD20:aCD37 was around 1 :1. Antibody-functionalized IFs of a second batch, batch B, were synthesized with the following adaptations to the protocol described above to reduce IF aggregation: antibodies were washed against PBS instead of borate buffer, and DBCO-functionalized with 2 equivalents of DBCO-PEG4-NHS ester (Jena BioScience) and labelled with 1 equivalent of AZDye™ 647 NHS ester (VectorLabs) or ATTO 488 NHS ester (ATTO-TEC). In all experiments, an antibody concentration of 10 pg / ml was used for soluble aCD20 and aCD37, aCD20-IF, and aCD37-IF. For aCD20 / 37-IF and the combination of soluble aCD20 and aCD37, a concentration of 20 pg / ml was used.Table 1.

[0161]

[0162] Equivalents (eq) of antibody added are relative to the number of azide groups. A lower equivalent of antibody added, resulted in a lower antibody density on the IF. Here, 0.1 eq or 0.2 eq of DBCO-functionalized antibody resulted in around 2 or 3 antibodies per IF, respectively.

[0163] 2Shown is the mean ± SD of duplicate measurements, assuming an equal distribution of antibodies over the PIC polymers.

[0164] Purity of IPs

[0165] The purity of the IFs was determined using SDS-PAGE. ~0.3 pg of IF or soluble antibody was run on Mini PROTEAN® TGX™ Precast Gels (Bio-Rad Laboratories) next to Precision Plus Protein™ All Blue Prestained Protein Standard marker (Bio-Rad Laboratories). Gels were scanned on a Typhoon Biomolecular Imager (Amersham), using a 635 nm and 488 nm laser to visualize the aCD20 and aCD37 signal, respectively. Antibodies conjugated to IFs cannot enter the gel and remain in the loading wells. Therefore, the purity of IFs was calculated by dividing the signal around 150 kDa (indicating unconjugated soluble aCD20) and 100 kDa (indicating unconjugated soluble aCD37) by the total signal.

[0166] Binding capacity of IFs

[0167] The binding capacity of the IFs was determined by flow cytometry. OCI-LY1 and OCI-LY19 cells were blocked in PBS containing 0.5% bovine serum albumin (BSA) and 0.1% sodium azide (PBA) supplemented with 2% human serum for 15 minutes at 4 °C. Cells were washed twice with PBS and seeded in a 96-well V-bottom plate at a density of 2x105- 5x105cells per well. Cells were stained with Zombie Violet viability dye (BioLegend, 1 :1000 in PBS) for 30 minutes at 4 °C. Cells were washed once with PBA and subsequently stained with 10 or 20 pg / ml IF or soluble antibody in PBA for 30 minutes on ice in the dark. Cells were washed twice with PBA before binding capacity of the IFs was measured by flow cytometry (FACS Lyric, BD Biosciences) using build-in Alexa Fluor 647 (AF647), Alexa Fluor 488 (AF488), and BD Horizon Brilliant Violet 421 (BV421) filter sets.

[0168] Immunofluorescence staining

[0169] Binding- and clustering capacity of the IFs was visualized by immunofluorescence staining. OCI-LY1 cells were put in fresh media the day before the experiment. OCI-LY1 cells were seeded inPBS in a 96-well V-bottom plate at a density of 5x105cells / well. Cells were resuspended in block buffer (3% bovine serum albumin and 2% human serum in PBS, filtered with a 0.45 pm syringe filter) and blocked on ice for 30 minutes. Cells were resuspended in RPMI 1640 media without phenol red (Gibco) supplemented with 1% stable glutamine (hereafter termed serum-free media (SFM)), containing 10 or 20 pg / ml IF, soluble antibody, or human lgG1 isotype (clone QA16A12, Biolegend). Cells were kept on ice in the dark for 30 minutes with subsequent incubation at 37 °C in a 5% CO2 incubator for 30 minutes. Cells were washed three times with PBS and were adhered on poly L-lysine (Sigma-Aldrich)-coated, Hellmanex (Sigma-Aldrich)-cleaned coverslips by incubation in a humidified chamber at 37 °C for 20 minutes. Cells were fixed in 4% paraformaldehyde (Sigma-Aldrich) for 20 minutes at room temperature. After fixation, cells were washed three times with PBS and incubated with 4’-6’-diamidino-2-phenylindole (DAPI, Sigma-Aldrich, 1 :3000 in PBS) for 5 minutes at room temperature in the dark to stain cell nuclei. Cells were washed once in PBS and once in phosphate buffer (0.1 M, pH 7.3) before coverslips were mounted on Superfrost™ plus adhesion microscope slides (Epredia) with Prolong Glass Antifade Mountant (ThermoFisher Scientific). Cell imaging was performed using a Zeiss LSM900 confocal microscope with Airyscan detector using a Plan-Apochromat 63x / 1.4 DIC M7 oil objective. Images were acquired and processed using ZEN software.

[0170] Cytotoxic capacity of IFs

[0171] The cytotoxic capacity of the IFs was determined using CDC and direct cell death assays. Heat-inactivated pooled human serum and FBS mentioned below were obtained by incubating the solutions for 30 minutes at 60 °C prior to experiments. Pooled human serum was retrieved from four different donors.

[0172] To determine CDC, OCI-LY1 cells were put in fresh media the day before the experiment. OCI-LY1 cells were seeded in serum-free medium (SFM) in a 96-well V-bottom plate at a density of 1x105cells / well. Cells were treated with SFM containing 20 or 40 pg / ml IF, soluble antibody, or human lgG1 isotype (clone QA16A12, Biolegend) for 30 minutes at 37 °C in a 5% CO2 incubator. After 30 minutes, 80% heat-inactivated pooled human serum or 80% active pooled human serum in SFM was added to the cells (final serum concentration 40% and IF or antibody concentration 10 or 20 pg / ml) and cells were incubated for 3 hours at 37 °C in a 5% CO2 incubator. Cells were washed once with PBS before cells were stained with Zombie Violet viability dye (BioLegend, 1 :1000 in PBS). Cells were washed once with PBS + 0.05% BSA before cell viability was assessed by flow cytometry (FACSVerse, BD Biosciences) using the build-in BV421 filter set.

[0173] To determine direct cell death, OCI-LY1 and OCI-LY19 cells were put in fresh media the day before the experiment. OCI-LY1 and OCI LY19 cells were seeded in SFM in a 96-well round-bottom plate at a density of 2x105cells / well. Cells were treated with SFM containing 10% heat-inactivated FBS and 10 or 20 pg / ml IF, soluble antibody, obinutuzumab (Gazyva, Roche) or human lgG1 isotype (clone QA16A12, Biolegend) for 24 hours at 37 °C in a 5% CO2 incubator. Cells were washed oncewith PBS before cells were stained with Zombie Violet viability dye (BioLegend, 1 :1000 in PBS). Cells were washed twice with PBS + 0.05% BSA before cell viability was assessed by flow cytometry (FACSLyric, BD Biosciences) using the build-in BV421 filter set. To quantify apoptosis, cells were washed once in Annexin V binding buffer (BioLegend) after Zombie Violet staining and subsequently stained with Annexin V PE-Cyanine 7 (BioLegend, 1 :24 in Annexin V binding buffer). Cells were not washed prior to analysis by flow cytometry (FACSLyric, BD BioSciences) using the build-in PE-Cy7 filter set.

[0174] Phospho flow cytometry

[0175] Phospho flow cytometry of phosphorylated p38 (p-p38) expression was performed to identify the mechanism involved in direct cell death induced by aCD20-IF. OCI-LY1 cells were put in fresh media the day before the experiment. OCI-LY1 cells were seeded in SFM in a 96-well round-bottom plate at a density of 2x105cells / well. Cells were incubated with 10 pM SB203850 (Cell Signaling Technology) or 0.001% DMSO (ThermoFisher Scientific) in SFM containing 10% heat-inactivated FBS for 1 hour at 37 °C in a 5% CO2 incubator. After 1 hour incubation, SFM containing 10% heat-inactivated FBS and 20 pg / ml aCD20-IF, soluble aCD20, or human-lgG1 isotype (clone QA16A12, Biolegend) was added to the cells (final IF or antibody concentration 10 pg / ml) and cells weresubsequently incubated for 4 hours at 37 °C in a 5% CO2 incubator. During the last 15 minutes of incubation, cells were stained with Zombie Violet viability dye (BioLegend, 1 :1000 in SFM containing 10% heat-inactivated FBS). After viability staining, cells were fixed immediately using the Foxp3 / transcription factor staining buffer set (ThermoFisher Scientific) for 10 minutes at 37 °C. Cells were washed twice with permeabilization buffer from the Foxp3 / transcription factor staining buffer set (ThermoFisher Scientific) and stored at 4 °C overnight. The next day, Fc receptors were blocked with Human TruStain FcX (BioLegend, 1 :200 in permeabilization buffer) for 15 minutes and cells were subsequently stained with rabbit, Cell Signaling Technology, 1 :100 in permeabilization buffer) for 30 minutes in the dark at room temperature. Cells were washed once and stained with Phycoerythrin(PE)-conjugated donkey anti-rabbit (Jackson ImmunoResearch, 1 :400 in permeabilization buffer) for 15 minutes in the dark at room temperature. Cells were washed once in permeabilization buffer before analysis by flow cytometry (FACS Verse, BD Biosciences) using the build in PE and BV421 filter sets.

[0176] Intracellular calcium assay

[0177] An intracellular calcium (Ca2+) assay was performed to identify the mechanism involved in direct cell death induced by aCD20-IF. OCI-LY1 cells were put in fresh media the day before the experiment. OCI-LY1 cells were seeded in SFM in 96-well round-bottom plates at a density of 2x105cells / well. Cells were treated with SFM containing 10% heat-inactivated FBS and 10 pg / ml aCD20-IF, soluble aCD20, or human-lgG1 isotype (clone QA16A12, Biolegend) for 4 hours at 37 °C in a 5% CO2 incubator. After 4-hour incubation, cells were washed once in SFM and subsequently incubated with 5 pM Fluo-4-AM (ThermoFisher Scientific) in SFM for 30 minutes at 37 °C in a 5% CO2 incubator to allow measurement of intracellular Ca2+. Cells were washed once in SFM andsubsequently incubated in SFM for 20 minutes at 37 °C in a 5% CO2 incubator to enable deesterification of AM esters located intracellularly, allowing Fluo-4 fluorescence. Cells were resuspended in PBS and analyzed immediately by flow cytometry (FACS Verse or FACS Lyric, BD BioSciences) using the build-in AF488 filter set.

[0178] Data analysis

[0179] Flow cytometry data was analyzed using FlowJo 10 software. For OCI-LY1 and OCI-LY19 cell binding assays, the following gating strategy was employed. Dead cells were excluded using the forward (FSC) versus sideward scatter (SSC) and by gating on Zombie Violet (BV421) negative cells. Single cells were then selected based on the FSC-A versus FSC-H plot. The gating strategy for cell death and intracellular Ca2+assays was to solely gate out debris to enable representative killing percentages and intracellular Ca2+levels, respectively. For phospho flow cytometry, no gates were set as no cell debris was visible. Gates were kept equal for all conditions within one experiment. Image analysis was performed in Fiji Imaged. Brightness and contrast settings of Airyscan confocal images were adjusted based on the signal visible after cells were treated with the soluble antibody and propagated to images of cells treated with corresponding IFs. CD20 clustering was quantified using a macro. In brief, cells were selected manually, excluding large IF aggregates, and saved as separate images. The macro was then used to automatically find maxima, perform segmentation, and apply Otsu thresholding. The cluster size was subsequently determined using the analyze particles plug-in. Statistical analyses were performed in GraphPad Prism 8. Data are displayed as mean ± SEM. Differences between conditions were tested via unpaired t-test or one- or two-way ANOVA, using Tukey’s or Bonferroni test for multiple comparisons as mentioned in the figure legends. A p-value of <0.05 was considered statistically significant.

[0180] Example 3. Results

[0181] Purity and binding capacity of aCD20-, aCD37-, and aCD20 / 37-IFs

[0182] Rituximab and otlertuzumab were functionalized with a DBCO group and fluorescently labeled with AZ647 or Atto488 to synthesize soluble aCD20 and aCD37, respectively. These modifications did not alter the molecular weights of the antibodies (data not shown). Soluble antibodies were subsequently attached to unconjugated (blank) IFs using click chemistry, yielding aCD20-, aCD37, and aCD20 / 37-IFs. After synthesis, IFs were tested for purity using SDS-PAGE. IFs were of high purity (> 95%, Fig. 1 A), meaning that a low amount of unconjugated soluble antibody was present in the samples.

[0183] Flow cytometry showed that IFs were about 5 to 10 times less efficient in binding to OCI-LY1 cells than the corresponding soluble antibodies (Fig. 1 B, Fig. 1 C). Binding of IFs was specific to CD20+and CD37+cells as OCI-LY19 cells, lacking CD20 and CD37 expression, did not display an increase in fluorescent signal after treatment with IFs compared to untreated cells. Binding of soluble antibodies and IFs to OCI-LY1 cells was subsequently visualized using immunofluorescencestaining (Fig. 1D).

[0184] aCD20-, aCD37-, and aCD20 / 37-IFs induce more direct cell death than corresponding soluble antibodies

[0185] Filtered IFs were evaluated on their cytotoxic capacity in vitro using CDC and direct cell death assays in OCI-LY1 cells. In contrast to soluble aCD20 and soluble aCD20 and aCD37 combined, IFs did not appear to induce cell death of OCI-LY1 cells through CDC, as no increase in IF-induced cell death was observed after incubation in active human serum compared to heat-inactivated human serum (Fig. 2A). Synergy between soluble aCD20 and aCD37 in inducing CDC, indicating an increase in cell death after incubation with soluble aCD20 and aCD37 combined compared to soluble aCD20 alone in active human serum, was observed in one out of two experiments. Surprisingly, cell death occurred both when IF-treated cells were incubated in heat-inactivated human serum, as well as active human serum (Fig. 2A). Thus, even though IFs did not appear to induce CDC, the IFs induced direct cell death regardless of the presence of active human serum.

[0186] To investigate whether IFs would induce more direct cell death than soluble aCD20 and aCD37, OCI-LY1 cells were incubated with IFs for 24 hours to enable full activation of apoptosis pathways. IFs induced significantly more direct cell death than the corresponding soluble antibodies (Fig. 2B). Furthermore, the aCD20 / 37-IF induced more direct cell death than the aCD20-IFs and aCD37-IFs. A higher antibody density on the aCD20-IF (0.2 eq versus 0.1 eq aCD20, see Example 1 , IF synthesis) did not increase direct cell death. Next to rituximab, another anti-CD20 antibody, obinutuzumab (OBZ), was taken along since OBZ is known to induce more direct cell death than rituximab (Awasthi, A., et al., 2015, doi:10.1111 / bjh.13764). In all cases, IFs showed higher levels of direct cell death than OBZ (all p< 0.05). Direct cell death induced by IFs was not a consequence of possible cytotoxic properties of the IF itself as a blank IF (bIF) did not induce direct cell death (Fig. 2B). Additionally, direct cell death induced by IFs was specific to CD20+and CD37+cells, as IFs did not induce direct cell death in OCI-LY19 cells.

[0187] Improving binding efficiency of aCD20-IF does not lead to an increase in direct cell death

[0188] A new batch of aCD20-IFs (batch B) was synthesized with a slightly adapted protocol to lower the amount of IF aggregates with the aim of improving IF binding efficiency. The SDS-PAGE gel revealed high purity of the newly synthesized aCD20-IF-B (97%, Fig. 3A). Flow cytometry showed that the mean binding efficiency of aCD20 IF-B to OCI-LY1 cells was around three times higher than that of batch A filtered aCD20-IF with similar antibody spacing (aCD20-IF (0.2 eq); aCD20-IF-A), however, statistical significance of this effect could not be determined as the binding efficiency of the two IFs was determined in separate experiments (Fig. 1C, Fig. 3B). Improved binding efficiency of aCD20-IF-B was also evident on immunofluorescence staining of OCI-LY1 cells, as almost all cells were labelled with similar fluorescence intensity compared to soluble aCD20, and IF aggregates were largely absent (Fig. 3C). Binding of aCD20 IF-B was specific to CD20+cells, asOCI-LY19 cells did not display an increase in fluorescent signal after treatment with aCD20-IF-B compared to cells treated with an isotype control antibody. Direct cell death induced by aCD20-IF-A and aCD20-IF-B in OCI-LY1 cells was similar (Fig. 4A). The percentage of apoptotic OCI-LY1 cells was also comparable between the two IFs (Fig. 4B). However, when using a non-saturating concentration of IF, a trend was observed towards higher direct cell death with aCD20-IF-B compared to aCD20-IF-A in OCI-LY1 cells (Fig. 4C). Direct cell death induced by aCD20-IF-B was absent in OCI-LY19 cells, indicating that the induction of direct cell death was specific to CD20+and CD37+cells. Similarly to aCD20 IF-A, aCD20-IF-B did not induce CDC in OCI-LY1 cells.

[0189] aCD20-IF upregulates p-p38 and intracellular Ca2+levels compared to soluble aCD20 Phosphorylated p38 (p-p38) expression levels and intracellular Ca2+levels were previously shown to be implicated in direct cell death induced by rituximab once CD20 clustering was induced by rituximab crosslinking. Thus, to indirectly investigate CD20 clustering, p-p38 expression levels and intracellular Ca2+levels were investigated after OCI-LY1 cells were incubated with either soluble aCD20 or aCD20 IF-B. Phospho flow cytometry showed a significant upregulation of p-p38 when cells were incubated with aCD20-IF-B compared to soluble aCD20. Direct cell death induced by aCD20-IF-B was dependent on the upregulation of p-p38 (Fig. 5A), since addition of the p38 inhibitor SB203580 significantly decreased cell death (Fig. 5B). This suggests an important role for p-p38 in the induction of direct cell death by aCD20-IF-B. Flow cytometry displayed that aCD20-IF-B induced around 1.5 times higher intracellular Ca2+levels than soluble aCD20 (Fig. 5D), using Fluo-4-AM as an indicator of intracellular Ca2+(Fig. 5C)

[0190] Thus, this study shows that tumor cell cytotoxicity in DLBCL can be increased through simultaneous targeting of CD20 and CD37 by combining rituximab (aCD20) and otlertuzumab (aCD37) on IFs. It is shown that aCD20-, aCD37-, and aCD20 / 37-IFs were more potent in inducing direct cell death than soluble aCD20 and aCD37, and a trend towards higher direct cell death was observed with the aCD20 / 37-IF compared to aCD20- and aCD37-IFs, suggesting a synergistic effect. The cell death induced by the aCD20-, aCD37-, and aCD20 / 37-IFs appeared to be independent of CDC. Furthermore, an upregulation of the rituximab-associated apoptotic markers p-p38 and intracellular Ca2+was found after treatment with aCD20 IF compared to soluble aCD20, which are markers known to be implicated once CD20 clustering is induced. Thus, the finding that aCD20 / 37-IFs are more potent in inducing direct cell death than soluble aCD20 and aCD37, is possibly related to increased CD20 and CD37 clustering on the cell membrane. Overall, the aCD20 / 37-IFs could be an effective immunotherapy for treating DLBCL.

[0191] Example 4. Therapeutic potential of nanosized immunofilaments in hematological malignancies 4.1 Abstract

[0192] CD38 is a well-established therapeutic target in multiple myeloma and B cell lymphoma, commonly targeted by monoclonal antibodies such as daratumumab and isatuximab. However, thesetherapies often fail to induce durable responses, emphasizing the need for improved strategies. Here we present a versatile therapeutic platform based on conjugating daratumumab and isatuximab to semiflexible immunofilaments (IFs). We show that CD38-targeting IFs mediate improved tumor cell killing compared to soluble daratumumab and isatuximab through enhanced complement-dependent and direct cytotoxicity. To confirm the versatility of this approach, we developed IFs targeting CD20 using the monoclonal antibody rituximab, the standard of care for different B-cell non-Hodgkin lymphomas. CD20-targeting IFs also displayed increased direct cytotoxicity in lymphoma cells compared to rituximab. These results show that nanosized IFs induce cytotoxicity across various B cell malignancies, supporting IF-conjugated antibodies as a promising therapeutic strategy for hematological cancers.

[0193] 4.2 Introduction

[0194] Multiple myeloma (MM) is the second most common type of hematological cancer in adults and is characterized by the abnormal proliferation and accumulation of malignant plasma cells in the bone marrow. CD38 is a type II transmembrane glycoprotein that is highly expressed on malignant plasma and mature B cells, making it an important clinical target for hematological cancers including MM and some types of lymphoma and leukemia. CD38 functions both as an ectoenzyme and as a receptor that contributes to disease progression of MM through multiple mechanisms. For instance, it converts NAD+ into cyclic ADP-ribose (cADPR) and NADP+ into nicotinic acid adenine dinucleotide phosphate (NAADP), both of which can mobilize intracellular calcium. CD38 also mediates MM cell adhesion to endothelial cells via interaction with its ligand CD31 , facilitating MM retention in the bone marrow and supporting their survival. Additionally, its enzymatic activity leads to the production of adenosine, which induces a more immunosuppressive bone marrow microenvironment. Daratumumab, a monoclonal antibody targeting CD38, is the primary therapeutic agent used to treat both newly diagnosed and relapsed / refractory MM. Daratumumab binding to CD38 on MM cells can induce tumor cell death through different mechanisms, including complement-dependent cytotoxicity (CDC), and antibody-dependent cellular phagocytosis / cytotoxicity (ADCP, ADCC). Additionally, daratumumab can induce programmed cell death when crosslinked through its Fc region, and can deplete CD38-positive immune suppressor cells, including regulatory T cells / B cells and myeloid-derived suppressor cells. Daratumumab can also block the enzymatic and cell adhesion functions of CD38, thereby inhibiting MM survival. Isatuximab is another FDA-approved monoclonal antibody targeting CD38, which shares cytotoxic mechanisms with daratumumab, including CDC, ADCC, and ADCP. Unlike daratumumab, isatuximab can directly induce apoptosis in MM cells without crosslinking, and more effectively inhibits CD38 enzymatic activity by binding near the active site, while daratumumab affects CD38 function indirectly, likely through conformational changes. Daratumumab and isatuximab are also under clinical investigation for the treatment of B cell lymphoma, given that lymphoma cells can express high CD38 levels.

[0195] Although antibody-based therapies have advanced the treatment of hematological malignancies,many patients do not respond, and disease progression eventually occurs due to acquired resistance in MM cells (I. Saltarella et al., 2020, doi: 10.3390 / cells9010167). Current nanomedicine strategies such as nanoparticle-based systems aim to improve antibody efficacy by enabling receptor crosslinking, targeted delivery, reduced toxicity, and / or immune modulation, thereby overcoming resistance mechanisms and broadening therapeutic options. However, their clinical translation is often limited by heterogeneous tumor penetration, variable immune clearance or biodistribution, and complex manufacturing processes that can affect scalability and reproducibility (K.M. Abdullah, 2025, doi: 10.1186 / s12943-025-02368-w). Here we demonstrate a versatile therapy platform against diseases such as hematological malignancies, based on the coupling of therapeutic antibodies to semiflexible nanosized immunofilaments (IFs). These antibody-decorated scaffolds retain the functional specificity of monoclonal antibodies while presenting them in a multivalent format. Compared to bulkier nanocarriers, IFs represent a simpler, more effective and versatile alternative, capable of overcoming delivery and manufacturing limitations associated with conventional nanoparticles. Moreover, they offer the possibility to incorporate different antibodies or additional components such as cytokines, chemokines or cytotoxic drugs, aiming to enhance anti-tumor immune responses, prevent immune escape and / or promote direct tumor cell killing. Previous studies have shown that IFs conjugated with aCD3 and aCD28 significantly enhance T cell activation compared to soluble aCD3 and aCD28. IFs functionalized with pMHC and IL-2 further support antigen-specific T cell expansion and reduce tumor growth, and IFs presenting IL-12 and aCD16 potentiate NK cell activation and IFNy production. Here we designed IFs to target tumor cells by conjugating different therapeutic antibodies and evaluated their efficacy in models of hematological cancers, such as multiple myeloma and B cell lymphoma models.

[0196] 4.3 Results and discussion

[0197] CD38-targeting IFs: Daratumumab and Isatuximab were fluorescently labeled with AZDye 647 (AZ647) for imaging purposes and functionalized with a DBCO group to allow subsequent attachment to the IF using bioorthogonal click chemistry (Fig. 6A) through reactive azide monomers (N3) present in the side chains of the IF. The IF features a semiflexibile and water soluble helical polyisocyanopeptide (PIC) polymer with an average length of 400 nm. An average of 6 (5.6 ± 0.2, mean ± SD ) and 8 (8.3 ± 0.2, mean ± SD) antibodies per IF were attached to generate daratumumab- and isatuximab-IFs, respectively, quantified by dividing the average PIC length by the spacing (in nm) between adjacent antibodies coupled to the IF: 70.5 ± 2.1 nm (mean ± SD) for daratumumab and 48 ± 0 nm (mean ± SD) for isatuximab. For all experiments, soluble antibodies (labeled with AZ647) were used as controls for IF-conjugated antibodies, and the same antibody concentration was used for both conditions.

[0198] Binding of CD38-targeting IFs to CD38-expressing cells: we analyzed the capacity of the generated daratumumab- and isatuximab-IFs to bind CD38 expressed by different tumor cell lines using flow cytometry and confocal microscopy. First, the optimal antibody concentration was determined by performing a binding titration with daratumumab- and isatuximab-IFs. Saturation was reached at5 |jg / ml, which was used for all subsequent experiments. IF-conjugated antibodies were overall very efficient in binding CD38 in RPMI-8226, LP-1 , and DAUDI cell lines (Fig. 6B, 6B-D), which was further validated by binding to primary MM cells (Fig. 6E). As control, CD38-targeting IFs did not bind to the CD38-negative KM-H2 cell line (Fig. 6F). We next investigated whether conjugation to IFs altered surface CD38 availability. Internalization of antibody-CD38 complexes was minimal in RPMI-8226, LP-1 and DAUDI cells treated with daratumumab- or isatuximab-IFs or the corresponding soluble antibodies (Fig. 6G-I). Overall, our data indicate that IF-conjugated antibodies retain their capacity to bind CD38 with minimal internalization, thereby remaining accessible at the cell surface to exert their cytotoxic effects.

[0199] Induction of CDC by CD38-targeting IFs: next, we studied the tumor killing capacity of daratumumab- and isatuximab-IFs, starting with assessing their capacity to induce CDC. CDC is a mechanism of complement-mediated cell killing in which antibodies bound to surface antigens recruit the complement protein C1q. This event initiates the classical complement pathway, a proteolytic cascade that leads to the formation of the membrane attack complex, which creates a pore at the cell membrane, inducing cell lysis. The CDC-sensitive cell lines LP-1 (MM) and DAUDI (B cell lymphoma) were analyzed for cell death (propidium iodide +) after incubation with daratumumab-IFs or isatuximab-IFs, or their corresponding soluble antibodies, in the presence of human serum. Daratumumab-IF induced significantly higher CDC than soluble daratumumab in both cell lines (Fig. 7A-B). Isatuximab-IF was significantly more effective in inducing CDC than soluble isatuximab in DAUDI cells, with a similar trend observed in LP-1 cells (Fig. 7A-B, right panels). Isotype antibodies were used as negative control, and experiments with heat-inactivated serum that does not contain active complement confirmed CDC-specificity. Validating the specificity of CD38-targeting IFs in inducing CDC, treatment of LP-1 and DAUDI cells with control IF without antibody-conjugation did not induce cell death (Fig. 7C-D). In addition, the viability of the CDC-resistant RPMI-8226 cell line and the CD38-negative KM-H2 cell line were not affected by the CD38-targeting IFs as expected. CDC requires the formation of hexameric IgG-Fc complexes at the cell surface that facilitates C1q binding and subsequent initiation of the complement cascade. We hypothesized that the enhanced CDC effect observed with the IFs was due to increased CD38 receptor clustering. We tested this hypothesis by quantifying the number and size of CD38 clusters in DAUDI cells upon treatment with daratumumab- or isatuximab-IFs or the corresponding soluble antibodies. Although no significant differences in CD38 cluster formation between IFs and soluble antibodies were observed by quantifying confocal microscopy images (Fig. 8A-D), follow-up investigation using super-resolution microscopy, such as STORM, could be used to confirm or exclude nanoscale differences in CD38 organization.

[0200] Induction of ADCC by CD38-targeting IFs: we next evaluated whether daratumumab-IF could induce ADCC in the CDC-resistant RPMI-8226 MM cell line, using natural killer (NK) cells as effector cells. Daratumumab-IF triggered stronger ADCC than the soluble antibody, as shown by the increased MM cell death (eF780 +) upon incubation with NK cells. Isotype antibodies were used asnegative control (Fig. 9).

[0201] Induction of direct cytotoxicity by CD38-targeting IFs'. we evaluated the ability of daratumumab- and isatuximab-IFs to induce direct cytotoxicity using the RPMI-8226 cell line model. Cell death was quantified by measuring the percentage of Annexin V-PE + (early apoptotic) and eF450 + (late apoptotic or necrotic) cells after incubation with daratumumab-IFs or isatuximab-IFs or their corresponding soluble antibodies, or control IF. The gating strategy is shown in Fig. 10A. Daratumumab-IF was more potent than the soluble daratumumab antibody in promoting direct cell death in RPMI-8226 cells after 24 h incubation, demonstrated by increased levels of early and late apoptotic as well as necrotic cells (Fig. 10B), suggesting that IF-conjugation facilitates antibody crosslinking upon target binding. A similar trend was observed after 6 h incubation (Fig. 10B). In LP-1 and DAUDI cell lines, daratumumab-IF did not induce significant changes in the percentage of apoptotic cells compared to the soluble antibody (Fig. 10C-D), in line with studies reporting that daratumumab does not induce direct cell death. Isatuximab-IF was more potent than the soluble antibody in promoting direct cell death in RPMI-8226 cells both after 24 h (increased levels of early and late apoptotic as well as necrotic cells) and 6 h (increased levels of late apoptotic and necrotic cells), with a similar trend observed for early apoptotic cells at 6 h (Fig. 10E). Isatuximab-IF also significantly increased the percentage of late apoptotic cells in LP-1 and DAUDI cells after 6 h (LP-1 and DAUDI) or 24 h (LP-1) incubation compared to soluble antibodies (Fig. 10F-G), showing that isatuximab-IF can induce direct cytotoxicity across multiple CD38+ MM and lymphoma cell lines. Importantly, control IF did not induce cytotoxicity in any condition or cell model (Fig. 10B-G), confirming that the observed effects are mediated by the therapeutic antibody and not due to nonspecific cytotoxicity induced by the IF backbone.

[0202] Cleaved caspase-3 is a marker of apoptosis. To further confirm the induction of apoptosis upon daratumumab-IF or isatuximab-IF treatment, we quantified presence of cleaved caspase-3 in RPMI-8226 cells using flow cytometry. As shown, daratumumab-IFs and isatuximab-IFs increased caspase-3 cleavage compared to their soluble forms, confirming their improved pro-apoptotic activity in MM cell lines (Fig. 11 A). We next investigated whether CD38-targeting IFs influence intracellular calcium, a mediator of apoptosis and receptor crosslinking including CD38 and CD20. Increased intracellular calcium can drive mitochondrial depolarization and caspase-3 activation. We surprisingly found that daratumumab-IFs and isatuximab-IFs increased intracellular calcium levels compared to their soluble counterparts in LP-1 (Fig. 11 B), RPMI-8226 (Fig. 11 C) and DAUDI cells (Fig. 11 D), as measured using Fluo-4 AM.

[0203] Discussion on working mechanisms of CD38-targeting IFs: while the increase in intracellular calcium aligns with apoptosis induction, it may also reflect additional effects mediated by CD38 crosslinking, as daratumumab-IF elevated calcium in all cell lines without inducing significant apoptosis in LP-1 and DAUDI cells (Fig. 10C-D). The calcium increase observed here suggests that additional signaling mechanisms, such as receptor crosslinking, may be involved. CD38-targeting IFs may further disrupt tumor immune evasion by interfering with CD38-mediated adenosineproduction, potentially further enhancing its therapeutic efficacy. Moreover, CD38-targeting IFs may exert beneficial immunomodulatory effects by depleting CD38-expressing regulatory immune cells, such as regulatory T cells and myeloid-derived suppressor cells.

[0204] In addition to crosslinking, IF-conjugated antibodies may also enhance cytotoxicity through mechanotransduction, since their multivalency may apply strong mechanical forces upon receptor engagement. Several surface receptors, such as the T cell receptor, and integrins have been reported to act as mechanosensors. Although CD38 mechanosensory properties remain uncharacterized, it is plausible that IF-conjugated antibodies exert stronger mechanical forces upon target engagement than soluble antibodies, potentially enhancing downstream signaling and promoting cell death.

[0205] CD20-targeting IFs: CD20 is a major therapeutic target in B cell malignancies and autoimmune diseases, and the antibody rituximab is the standard of care in diffuse large B-cell lymphoma and other non-Hodgkin lymphomas. To investigate the versatility of tumor-targeting IFs in different therapeutic settings, we conjugated AZDye 647-labeled rituximab to IFs using bioorthogonal click chemistry and characterized their composition in terms of antibody spacing (in nm) and the number of antibodies per IF. The average antibody spacing was 136 nm ± 8.5 nm, and the number of antibodies per IF was 2.9 ± 0.2 based on duplicate measurements.

[0206] Binding of CD20-targeting IFs to CD20-expressing cells: a binding titration of rituximab-IF showed saturation at 10 pg / ml, which was used for all subsequent experiments (data not shown). Using this concentration, we compared binding of rituximab-IF and soluble rituximab to CD20 in three different lymphoma cell lines (OCI-LY1 , RAJI and DAUDI cells) by flow cytometry (Fig. 12A-C). As control, the CD20-negative OCI-LY19 cell line was included, which showed no rituximab-IF binding (Fig.

[0207] 12D). Following quantification of the flow cytometry data, we surprisingly found that rituximab-IFs has a lower binding capacity than soluble ritxumab, for all cell lines (Fig. 12E-G).

[0208] Induction ofCDC by CD20-targeting IFs: next, the ability of rituximab-IF to induce CDC was studied by measuring the percentage of dead cells (zombie violet +) treated with soluble rituximab or rituximab-IF in the presence of human serum or heat-inactivated serum controls. Rituximab-IF remained capable of inducing CDC in RAJI and DAUDI cells, though at lower levels than the soluble antibody (Fig. 13B-C), in contrast to OCI-LY1 cells that were already less sensitive to CDC by rituximab (Fig. 13A). We speculate that this limited CDC results from the low number of rituximab antibodies attached per IF (~3) due to decreased antibody spacing. This may hamper IgG hexamer formation, as several IFs would be required to form such a complex, unlike CD38-targeting IFs that carry a higher number of antibodies. Nonetheless, the strong direct cytotoxicity observed with rituximab-IF may provide a therapeutic advantage, particularly in contexts where CDC is inefficient, such as in the tumor microenvironment where complement is not available or inhibited, or when lymphoma cells upregulate the complement inhibitory proteins CD55 and CD59 to escape fromCDC. In line with CD38-targeting IFs, no differences were observed in CD20 cluster number or size in lymphoma cells treated with soluble rituximab or rituximab-IF (Fig. 14A-B).

[0209] Induction of direct cytotoxicity by CD20-targeting IFs: direct cytotoxicity induced by rituximab-IF or soluble rituximab antibody was analyzed in OCI-LY1 , RAJI and DAUDI cells by quantifying cell death (zombie violet +) using flow cytometry. Despite its lower binding capacity (Fig. 12), rituximab-IF was more potent at inducing direct cell death than soluble rituximab in all lymphoma cell lines, while the control IF did not induce any cell death as expected (Fig. 15A-C). These results confirm the specificity of rituximab-IF and support the feasibility of conjugating different therapeutic antibodies to IFs to enhance tumor cell killing.

[0210] To investigate the mechanism underlying the enhanced direct cytotoxicity induced by rituximab-IF, we analyzed calcium influx, as inducing calcium influx can trigger cell death. Additionally, we analyzed p38 phosphorylation, as p38 phosphorylation can lead to caspase-dependent apoptosis via phosphorylation of Bim. Rituximab-IF induced higher intracellular calcium levels detected by Fluo-4 AM (Fig. 16A), and increased p38 phosphorylation in OCI-LY1 cells compared to treatment with soluble rituximab (Fig. 16B). To validate the involvement of p38 signaling in rituximab-IF-induced apoptosis, pre-treatment with the p38 inhibitor SB203850 significantly reduced the percentage of p-p38 + cells and dead cells (zombie violet +) upon rituximab-IF incubation (Fig. 16B-C). These results indicate that rituximab-IF induces lymphoma cytotoxicity through enhanced calcium influx and p38 phosphorylation, which further supports the crosslinking capacity of the IFs.

[0211] Summary: we show that conjugating therapeutic antibodies to immunofilaments enhances their ability to kill multiple myeloma and B cell lymphoma cell lines compared to their soluble forms. Conjugation of daratumumab and isatuximab to IFs enhanced CDC and promoted direct cytotoxicity. Importantly, rituximab conjugation to the IF platform resulted in enhanced direct cytotoxicity of malignant B cell lines via elevated calcium influx and p38 phosphorylation. We note that co-targeting CD20 and CD38 with a dual-targeting IF design results in synergistic cytotoxicity of malignant B cells compared to their individual targeting effects while preserving Fc functions, offering a more effective therapeutic strategy with reduced likelihood of antigen escape. Importantly, IFs have been shown to be safe and well-tolerated in vivo supporting their validation as a promising therapeutic platform for cancer. Overall, we provide a versatile immunotherapy strategy with the potential to increase the efficacy of therapeutic antibodies across hematological malignancies.

[0212] 4.4 Materials and Methods

[0213] Isolation of malignant plasma cells from bone marrow or pleural fluid of multiple myeloma patients : malignant plasma cells were extracted from the bone marrow or pleural fluid of patients diagnosed with multiple myeloma. Bone marrow extracts were suspended in PBS, filtered through a 70pm filter and washed in PBS. Red blood cells (RBCs) were lysed using NH4CI (BD Pharmigen) diluted 1 :10 in demi-water for 15 min at room temperature (RT). Pleural fluid samples were processed using thesame RBC lysis protocol but without the filtration step.

[0214] Isolation of primary NK cells from peripheral blood: human peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats obtained from healthy volunteers. NK cells were isolated from peripheral blood using human NK cell magnetic-activated cell sorting isolation kit (130-092-657, Miltenyi) according to the manufacturer’s protocol.

[0215] Culture of Multiple Myeloma and lymphoma cell lines: human multiple myeloma LP-1 (Cat: ACC 41) and RPMI-8226 and lymphoma DAUDI, OCI-LY1 (Cat: ACC 722), RAJI (Cat: ACC 319), OCI-LY19 (Cat: ACC 528) and KM-H2 (Cat: ACC 8) cells were maintained at 37 °C in 5% CO2. Cells were cultured in either IMDM (Gibco) (LP-1) or RPMI 1640 (Gibco) (RPMI-8226, DAUDI, OCI-LY1 , RAJI and OCI-LY19) supplemented with 10% fetal bovine serum (FBS; Hyclone), 1% stable-glutamine (Capricorn Scientific) and 1% antibiotic-antimycotic (Gibco, Thermo Fisher Scientific). Cell lines were acquired from DSMZ (LP-1 , OCI-LY1 , RAJI, OCI-LY19 and KM-H2). RPMI-8226, DAUDI and KM-H2 lines were a kind gift from Dr. Blanca Scheijen (Dept. Pathology, Radboudumc). All cell lines were authenticated using STR analysis and routinely tested for mycoplasma.

[0216] Immunofilament (IF) synthesis: isocyanopeptide monomers with an azide- or methoxy end group were polymerized in a 1 :30 ratio, respectively, yielding PICs of -400 nm. To functionalize the antibodies, CD38 (daratumumab (Selleck) and isatuximab (MedChem)) and CD20 (rituximab (Mabthera, Roche)) antibodies were washed with borate buffer (pH 8.5, 50 mM: daratumumab, isatuximab) or phosphate-buffered saline (PBS) (pH 7.4: rituximab) using Amicon ultra centrifugal filters (30 kDa, Merck). Next, 1 equivalent of DBCO-PEG4-NHS ester (Jena BioScience), and 1.5 equivalents of AZDyeTM 647-NHS ester (VectorLabs), daratumumab, isatuximab or rituximab were added. After 2 h of reaction at 4 °C, the antibodies were purified using Zebaspin columns (Thermo Fisher Scientific, 7 Kda MWCO, 5 ml). The columns were prewashed 6 times with PBS before the reaction mixtures were loaded.

[0217] To prepare the antibody-IFs, 0.3 (daratumumab), 0.4 (isatuximab), 0.2 (rituximab) equivalents (relative to azide groups) of functionalized antibodies were added to the PIC and rotated for 18-22 h. After the indicated reaction time, samples were blocked with azido-PEG3-amine (Conju-Probe, 2 pL per 100 pg PIC). The bioconjugates were purified in PBS using tangential flow filtration (TFF, Hansabiomed). Conjugated protein concentrations were determined using a SparkTM 10 M plate reader (Tecan), using the soluble antibodies as standards. PIC concentrations were determined using circular dichroism spectroscopy (JASCO J-810), after which the number of antibodies on the IFs could be calculated by dividing the average length of the PIC by the nm distance between antibodies on the PIC.

[0218] Flow cytometry: cell suspensions (2 x 105cells) were collected, washed in PBS and blocked with PBA (PBS + 1% bovine serum albumin (BSA) + 0.05% sodium azide) containing 2% human serum for 30 min at RT. Cells were then incubated with daratumumab-, isatuximab- or rituximab-IFs, ortheir corresponding soluble antibodies, at a concentration of 5 pg / ml (daratumumab and isatuximab) or 10 pg / ml (rituximab), for 30 min at 37 °C (RPMI-8226, LP-1 , DAUDI, RAJI, KM-H2 and primary MM cells) or 4 °C (OCI-LY1 and OCI-LY19). Cells were washed in PBS and analyzed on a FACSLyric flow cytometer (BD Biosciences). Data was analyzed using FlowJo X Software (FlowJo LLC).

[0219] Immunofluorescence microscopy: cell suspensions (2 x 105cells) were collected, washed in PBS, and blocked with PBS containing 3% BSA and 2% human serum for 30 min at RT. Next, cells were incubated with 5 pg / ml (daratumumab and isatuximab) or 10 pg / ml (rituximab) of the relevant soluble or IF-conjugated therapeutic antibodies for 30 min at 37 °C. Following this, cells were washed in PBS and adhered on Poly-L-lysine (PLL)-coated coverslips for 15-20 min at 37 °C. After seeding, cells were fixed in 4% paraformaldehyde (PFA) for 20 min at RT, washed in PBS and stained with 0.3 pg / ml 4’-6-diamidino-2-phenylindole (DAPI) for 5-10 min at RT. Cells were washed in PBS, followed by demineralized water (RPMI-8226, LP-1 , DAUDI and primary MM cells) or phosphate buffer (OCI-LY1), and subsequently embedded in Fluoromount G (Southern Biotech; RPMI-8226, LP-1 , DAUDI and primary MM cells) or ProLong™ Glass Antifade Mountant (Thermo Fisher Scientific; OCI-LY1). Confocal imaging was carried out using a Zeiss LSM900 microscope equipped with an Airyscan detector and a 63x oil immersion objective (NA 1.4). Microscope control, spectral detection adjustments for DAPI and other fluorescent markers, as well as Airyscan image processing, were performed using Zeiss Zen software. Image analysis was subsequently conducted with Fiji.

[0220] Internalization assay: cell suspensions (105cells) were collected and washed in RPMI 1640 serum-free media (SFM). Cells were incubated with 5 pg / ml daratumumab- or isatuximab-IFs, their corresponding soluble antibodies or with a human lgG1 ,k isotype control (Biolegend), in RPMI 1640 SFM for 30 min at 4 °C. Cells were then washed and incubated in RPMI 1640 SFM for 30 min, 1 , 2 or 3 h at 37 °C. After the incubation, remaining surface bound antibody was stripped by two rounds of washing the cells in low pH buffer (0.5 M NaCI, 0.5% acetic acid, pH 2.5-2.8 in demineralized water) for 2 min on ice, followed by neutralization with RPMI 1640 media. Cells were then washed in PBS and analyzed by flow cytometry. To distinguish internalized from surface-bound antibody signal, control samples were incubated with antibody-IFs at 4 °C for 3 h and were either stripped or left unstripped. Since no internalization occurs at 4 °C, the difference between the stripped and unstripped conditions at 4 °C represents the amount of signal at the cell surface. To calculate the % of antibody remaining on the cell surface after incubation at 37 °C, intracellular signal (signal after stripping) at each timepoint was subtracted from the total surface signal measured at 4 °C (unstripped). This difference was normalized to the total surface signal at 4 °C (the difference between unstripped and stripped controls at 4 °C), and multiplied by 100:

[0221] % surface signal = [(unstripped 4 °C - stripped 37 °C) / (unstripped 4 °C - stripped 4 °C)] x 100

[0222] Complement Dependent Cytotoxicity (CDC) assay: cell suspensions (105cells) were collected andwashed in RPMI 1640 SFM. Cells were incubated with 5 pg / ml (daratumumab and isatuximab) or 10 pg / ml (rituximab) soluble or IF-conjugated therapeutic antibodies, isotype control or control IF (no antibody conjugation, using the same PIC concentration), for 30 min at 37 °C. Cells were washed once in SFM (RPMI-8226, LP-1 , DAUDI and KM-H2 for CD38-targeting IFs) or left unwashed (OCI-LY1 , RAJI and DAUDI for CD20-targeting IF) and subsequently treated with 40% pooled human serum (Innovative Research) for 3 h at 37 °C. Heat-inactivated serum (30 min at 60 °C) was used as a control to confirm complement specificity, as this treatment inactivates complement proteins. After incubation, cells were washed with PBS and treated with 5 pg / ml Propidium Iodide (Miltenyi) for 10 min at RT (RPMI-8226, LP-1 , DAUDI and KM-H2 for CD38-targeting IFs) or incubated with 1 pg / ml zombie violet viability dye (Biolegend) for 20 min at RT and washed in PBS + 0.05% BSA (OCI-LY1 , RAJI and DAUDI for CD20-targeting IF). Cells were then washed in PBS and analyzed on a FACSLyric flow cytometer (BD Biosciences). Data was analyzed using FlowJo X Software (FlowJo LLC).

[0223] Direct Killing Assay: cell suspensions containing 105cells (RPMI-8226, LP-1 and DAUDI for CD38-targeting IFs) or 2 x 105(OCI-LY1 , RAJI and DAUDI for CD20-targeting IF) were collected and washed in RPMI 1640 media. Cells were incubated with 5 pg / ml (daratumumab and isatuximab) or 10 pg / ml (rituximab) soluble or IF-conjugated therapeutic antibodies, isotype control or control IF in RPMI 1640 media with 1% stable glutamine. The incubation was performed for 6 or 24 h at 37 °C. Afterwards, cells were washed once with PBS and incubated with 1 pg / ml eFluor 450 viability dye (Thermo Fisher Scientific; RPMI-8226, LP-1 and DAUDI for CD38-targeting IFs) or zombie violet viability dye (Biolegend; OCI-LY1 , RAJI and DAUDI for CD20-targeting IF) for 20 min at RT. To detect apoptotic cells, cells were washed with PBS and incubated with Annexin-PE using the PE Annexin V Apoptosis Detection Kit I (BD Biosciences) according to the manufacturer’s recommended protocol (RPMI-8226, LP-1 and DAUDI for CD38-targeting IFs) or directly washed in PBS + 0.05% BSA and measured by flow cytometry (OCI-LY1 , RAJI and DAUDI for CD20-targeting IF). To measure cleaved caspase-3 levels, cells were washed in PBS after the 6 h incubation, fixed in 4% PFA for 30 min at 4 °C, and permeabilized and blocked for 30 min at 4 °C in PBS containing 3% BSA, 2% human serum, 1% goat serum and 0.5% saponin. Cells were then stained in the same blocking buffer with an anti-cleaved caspase-3 antibody conjugated to AF488 (9669S, Cell Signaling Technology, 1 :20). Cells were washed in 0.5% saponin followed by PBS washes. All samples were measured using a FACSLyric flow cytometer (BD Biosciences) and data was analyzed using FlowJo X Software (FlowJo LLC).

[0224] Antibody-Dependent Cellular Cytotoxicity (ADCC) Assay: purified primary human NK cells were pre-activated with 1 ng / mL recombinant human IL-15 (Abeam) in RPMI 1640 media overnight. RPMI-8226 cells were labeled with CellTrace™ Violet (CTV, Thermo Fisher Scientific) following the manufacturer's protocol. Labeled cells were washed and plated in 96-well U-bottom plates (0.2*105cells / well). NK cells were added to the CTV-labeled RPMI-8226 cells at a 1 :1 effector-to-target (E:T) ratio, with 5 pg / ml soluble or IF-conjugated daratumumab, or isotype control, and incubated in RPMI1640 medium containing 1 ng / mL IL-15 (Abeam) and incubated with 5% CO2 for 16 h at 37 °C. After incubation, cells were washed and stained with 1 pg / ml eFluor 780 viability dye (Thermo Fisher Scientific) for 20 min at RT in the dark. Samples were washed and analyzed by flow cytometry on a FACSLyric flow cytometer (BD Biosciences). Data was analyzed using FlowJo X Software (FlowJo LLC).

[0225] Phosphoflow cytometry: cell suspensions containing 2 x 105OCI-LY1 cells were seeded in RPMI 1640 media and incubated with 10 pM SB203850 (Cell Signaling Technology) or 0.001% DMSO (Thermo Fisher Scientific) for 1 h at 37 °C. Cells were then treated with 10 pg / ml of rituximab in the soluble or IF form in RPMI 1640 media (Gibco) for 4 h. During the last 15 min, cells were stained with 1 pg / ml zombie violet viability dye (Biolegend), fixed and permeabilized using a Foxp3 / transcription factor staining buffer set (Thermo Fisher Scientific). Next, Fc receptors were blocked using Human TruStain FcX (Biolegend) and cells were stained with rabbit anti-phospho-p38 MAPK (Cell Signaling Technology, 1 :100), followed by donkey anti-rabbit PE-conjugated secondary antibody (Jackson ImmunoResearch, 1 :400). Cells were analyzed on a FACSVerse cytometer (BD Biosciences). Data was analyzed using FlowJo X Software (FlowJo LLC).

[0226] Measurement of intracellular Ca2+: cell suspensions (2 x 105cells) were treated with 5 pg / ml (daratumumab and isatuximab) or 10 pg / ml (rituximab) soluble or IF-conjugated daratumumab, isatuximab or rituximab or with isotype control, in RPMI 1640 media for 4 h at 37 °C. Cells were washed in RPMI 1640 SFM and subsequently incubated with 5 pM Fluo-4 AM (Thermo Fisher Scientific) in SFM for 30 min at 37 °C. Cells were washed and incubated in SFM for an additional 20 min at 37 °C, as a de-esterification step. Next, cells were resuspended in PBS and analyzed on a FACSVerse or FACSLyric flow cytometer (BD Biosciences). Data was analyzed using FlowJo X Software (FlowJo LLC).

[0227] Image and Statistical Analysis: confocal image analyses were performed using Fiji image analysis software. Background on fluorescent images was removed by adjusting the brightness & contrast settings. Cells were randomly selected prior to image analysis, excluding blebbing cells. For the CD38 and CD20 cluster analysis upon daratumumab, isatuximab or rituximab binding, local maxima were found, followed by segmentation of maxima. Otsu autothresholding was applied, and both segmented and thresholded images were combined using the image calculator. Analyze particles plug-in in Fiji software was used to determine the number and size of clusters. All statistical analysis was performed using GraphPad Prism 8 software, and data are expressed as mean ± SD, as indicated in the figure legends. Statistical tests performed are specified in the figure legends. Statistical significance was set at P < 0.05.

Claims

Claims1. A conjugate comprising a polyisocyanide polymer and a first binding moiety capable of binding a membrane protein of a cancer cell.

2. The conjugate according to claim 1 , wherein the binding moiety is an antibody or a fragment thereof.

3. The conjugate according to claim 1 or 2, wherein the number of first binding moieties per polyisocyanide polymer is between 1 to 10, preferably between 1 to 4, more preferably it is 2 or 3.

4. The conjugate according to any one of claims 1-3, wherein the first binding moiety is capable of binding CD20, CD38, CD45, CD5, CD19, CD10, CD30, CD7, CD2, CD23, CD43, CD103, CD13, CD33, CD1 a or CD22, preferably CD20, CD38, CD45, CD5, CD19, or CD10, more preferably CD20 or CD38, most preferably CD20.

5. The conjugate according to claim 4, wherein the first binding moiety is Ritixumab, Daratumumab, Isatuximab, Brentuximab vedotin, Epcoritamab, Glofitamab, Ibritumomab tiuxetan, Mogamulizumab, Obinutuzumab, Ocrelizumab, Ofatumumab, Polatuzumab vedotin, Ripertamab, Ublituximab, or Zuberitamab, preferably Ritixumab, Daratumumab, Isatuximab, Ibritumomab tiuxetan, Obinutuzumab, Ocrelizumab, Ofatumumab, Ripertamab, Ublituximab, or Zuberitamab, more preferably Ritixumab, Ibritumomab tiuxetan, Obinutuzumab, or Ofatumumab, most preferably Rituximab.

6. The conjugate according to any one of claims 1-3, wherein the first binding moiety is capable of binding a membrane protein of the tetraspanin superfamily, preferably CD37, CD9, CD53, CD63, CD81 , TSPAN13, TSPAN4, CD151 , or CD82, most preferably CD37.

7. The conjugate according to claim 6, wherein the first binding moiety is Otlertuzumab, Lilotomab, or Naratuximab, preferably Otlertuzumab.

8. The conjugate according to any one of claims 1-7, further comprising a second binding moiety capable of binding a membrane protein of a cancer cell.

9. The conjugate according to claim 8, wherein the total number of first and second binding moieties per polyisocyanide polymer is between 2 to 10, preferably between 2 to 4, more preferably it is 2 or 3.

10. The conjugate according to claim 8 or 9, wherein the conjugate comprises at least one first binding moiety and at least one second binding moiety,wherein the first binding moiety is capable of binding CD20, CD38, CD45, CD5, CD19, CD10, CD30, CD7, CD2, CD23, CD43, CD103, CD13, CD33, CD1a or CD22, preferably CD20, CD38, CD45, CD5, CD19, or CD10, more preferably CD20 or CD38, most preferably CD20;wherein the second binding moiety is capable of binding a membrane protein of the tetraspanin superfamily, preferably CD37, CD9, CD53, CD63, TSPAN13, TSPAN4, CD151 , CD81 , or CD82, most preferably CD37.

11. The conjugate according to any one of claims 1-10, wherein the polyisocyanide polymer has a length of about 100 to 1000 nm, preferably about 200 to 600 nm, more preferably about 400 nm.

12. The conjugate according to any one of claims 1-11 , wherein the cancer cell is a haematological cancer cell, preferably a lymphoma cell, a chronic lymphocytic leukemia cell, or a multiple myeloma cell, more preferably a non-Hodgkin lymphoma cell.

13. The conjugate according to any one of claims 1-12 for use as a medicament, wherein the medicament is preferably for treating cancer, more preferably haematological cancer such as lymphoma, chronic lymphocytic leukemia, or multiple myeloma, even more preferably non-Hodgkin lymphoma, most preferably diffuse large B cell lymphoma.

14. The conjugate for use according to claim 13, wherein the conjugate is for promoting p38 phosphorylation in the cancer cell.

15. Method for treating cancer in a subject, the method comprising the step of administering the conjugate according to any one of claims 1-12 to the subject.

16. The conjugate according to any one of claims 1-4, or 8-15, wherein the first binding moiety is capable of binding CD38.

17. The conjugate according to claim 16, wherein the first binding moiety is Daratumumab, Isatuximab, Felzartamab, Mezagitamab, HLX15, or CID-103, preferably Daratumumab, Isatuximab, Felzartamab, or Mezagitamab, more preferably Daratumumab or Isatuximab.

18. The conjugate according to claim 17, wherein the first binding moiety is Daratumumab.

19. The conjugate according to claim 17, wherein the first binding moiety is Isatuximab.

20. The conjugate according to any one of claims 16-19, wherein the number of first binding moieties per polyisocyanide polymer is between 1 to 20, preferably between 3 to 10, morepreferably between 5-9, most preferably between 6-8.

21. The conjugate according to any one of claims 1-4, or 8-15, wherein the first binding moiety is capable of binding CD20.

22. The conjugate according to claim 21 , wherein the first binding moiety is Rituximab, Ibritumomab tiuxetan, Obinutuzumab, Ocrelizumab, Ofatumumab, Ublituximab, Zuberitamab, Veltuzumab, Ocaratuzumab, or Tositumomab, preferably Rituximab, Ibritumomab tiuxetan, Obinutuzumab, Ocrelizumab, Ofatumumab, or Ublituximab, Zuberitamab.

23. The conjugate according to claim 22, wherein the first binding moiety is Rituximab.

24. The conjugate according to any one of claims 21-23, wherein the number of first binding moieties per polyisocyanide polymer is between 1 to 10, preferably between 1 to 4, more preferably it is 2 or 3.

25. The conjugate according to any one of claims 16-24, wherein the polyisocyanide polymer has a length of about 100 to 1000 nm, preferably about 200 to 600 nm, more preferably about 400 nm.

26. The conjugate according to any one of claims 16-25, wherein the cancer cell is a haematological cancer cell.

27. The conjugate according to claim 26, wherein the haematological cancer cell is a lymphoma cell, a leukemia cell, or a myeloma cell, preferably a lymphoma cell, a chronic lymphocytic leukemia cell, or a multiple myeloma cell.

28. The conjugate according to any one of claims 16-27 for use as a medicament.

29. The conjugate for use according to claim 28, wherein the medicament is for treating cancer, preferably haematological cancer, more preferably lymphoma, leukemia, or myeloma, most preferably lymphoma, chronic lymphocytic leukemia, or multiple myeloma.

30. The conjugate for use according to claim 28 or 29, wherein the conjugate is for promoting p38 phosphorylation and / or calcium influx in the cancer cell.

Images