Patent Application
20240409653
The present invention relates to combination therapy using a binding agent that binds to human PD-L1 and to human CD137 in combination with pembrolizumab to reduce or prevent progression of a tumor or treating cancer.
CD137 (4-1BB) is a member of the TNFR family and is a co-stimulatory molecule on CD8+ and CD4+ T cells, regulatory T cells (Tregs), Natural Killer T cells (NK(T) cells), B cells and neutrophils. On T cells, CD137 is not constitutively expressed, but induced upon T-cell receptor (TCR) activation (for example, on tumor infiltrating lymphocytes (TILs) (Gros et al., J. Clin Invest 2014; 124(5):2246-59)). Stimulation via its natural ligand 4-1BBL or agonist antibodies leads to signaling using TRAF-2 and TRAF-1 as adaptors. Early signaling by CD137 involves K-63 poly-ubiquitination reactions that ultimately result in activation of the nuclear factor (NF)-κB and mitogen-activated protein (MAP)-kinase pathways. Signaling leads to increased T cell co-stimulation, proliferation, cytokine production, maturation and prolonged CD8+ T-cell survival. Agonistic antibodies against CD137 have been shown to promote anti-tumor control by T cells in various pre-clinical models (Murillo et al., Clin Cancer Res 2008; 14(21):6895-906). Antibodies stimulating CD137 can induce survival and proliferation of T cells, thereby enhancing the anti-tumor immune response. Antibodies stimulating CD137 have been disclosed in the prior art, and include urelumab, a human IgG4 antibody (AU 2004279877) and utomilumab, a human IgG2 antibody (Fisher et al., 2012, Cancer Immunol. Immunother. 61: 1721-1733).
Programmed death ligand 1 (PD-L1, PDL1, CD274, B7H1) is a 33 kDa, single-pass type I membrane protein. Three isoforms of PD-L1 have been described, based on alternative splicing. PD-L1 belongs to the immunoglobulin (Ig) superfamily and contains one Ig-like C2-type domain and one Ig-like V-type domain. Freshly isolated T and B cells express negligible amounts of PD-L1 and a fraction (about 16%) of CD14+ monocytes constitutively express PD-L1. However, interferon-γ (IFNγ) is known to upregulate PD-L1 on tumor cells.
PD-L1 obstructs anti-tumor immunity by 1) tolerizing tumor-reactive T cells by binding to its receptor, programmed cell death protein 1 (PD-1) (CD279) on activated T cells; 2) rendering tumor cells resistant to CD8+ T cell and Fas ligand-mediated lysis by PD-1 signaling through tumor cell-expressed PD-L1; 3) tolerizing T cells by reverse signaling through T cell-expressed CD80 (B7.1); and 4) promoting the development and maintenance of induced T regulatory cells. PD-L1 is expressed in many human cancers, including melanoma, ovarian, lung and colon cancer (Latchman et al., 2004 Proc Natl Acad Sci USA 101, 10691-6).
PD-L1 blocking antibodies have shown clinical activity in several cancers known to overexpress PD-L1 (incl. melanoma, NSCLC). For example, atezolizumab is a humanized IgG1 monoclonal antibody against PD-L1. It is currently in clinical trials as an immunotherapy for several indications including various types of solid tumors (see e.g. Rittmeyer et al., 2017 Lancet 389:255-265) and is approved for non-small-cell lung cancer and bladder cancer indications. Avelumab, a PD-L1 antibody, (Kaufman et al Lancet Oncol. 2016; 17(10):1374-1385) has been approved by the FDA for the treatment of adults and pediatric patients 12 years and older with metastatic Merkel cell carcinoma, and is currently in clinical trials in several cancer indications, including bladder cancer, gastric cancer, head and neck cancer, mesothelioma, NSCLC, ovarian cancer and renal cancer. Durvalumab, a PD-L1 antibody, is approved for locally advanced or metastatic urothelial carcinoma indications, and is in clinical development in multiple solid tumors and blood cancers (see e.g. Massard et al., 2016 J Clin Oncol. 34(26):3119-25). Further anti-PD-L1 antibodies have been described e.g in WO2004004771.
Horton et al (J Immunother Cancer. 2015; 3(Suppl 2): 010) discloses combination of an agonistic 4-1BB antibody with a neutralizing PD-L1 antibody. WO 2019/025545 provides binding agents, such as bispecific antibodies, binding human PD-L1 and binding human CD137.
However, despite these advances in the art there is a considerable need for improved therapies to prevent progression of a tumor or treating cancer.
The present inventors have surprisingly found that a combination of (i) stimulation with a binding agent binding human PD-L1 and binding human CD137 and (ii) an antibody binding to Programmed Death-1 (PD-1) amplifies the immune response.
Thus, in a first aspect, the present disclosure provides a binding agent for use in a method for reducing or preventing progression of a tumor or treating cancer in a subject, said method comprising administering to said subject the binding agent prior to, simultaneously with, or after administration of an antibody binding to Programmed Death-1 (PD-1), or an antigen-binding fragment thereof, wherein the binding agent comprises a first binding region binding to CD137 and a second binding region binding to PD-L1;
In a second aspect, the present disclosure provides a kit comprising
In a third aspect, the present disclosure provides a kit of the second aspect for use in a method for reducing or preventing progression of a tumor or treating cancer in a subject.
In a fourth aspect, the present disclosure provides a method for reducing or preventing progression of a tumor or treating cancer in a subject, said method comprising administering to said subject a binding agent comprising a first binding region binding to CD137 and a second binding region binding to PD-L1
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Although the present disclosure is further described in more detail below, it is to be understood that this disclosure is not limited to the particular methodologies, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present disclosure which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.
In the following, the elements of the present disclosure will be described in more detail. These elements are listed with specific embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and preferred embodiments should not be construed to limit the present disclosure to only the explicitly described embodiments. This description should be understood to support and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed and/or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application unless the context indicates otherwise. For example, if in a preferred embodiment of the binding agent used herein the first heavy chain comprises or consists essentially of or consists of an amino acid sequence set forth in SEQ ID NO: 23 or 29 [IgG1-Fc_FEAR] and in another preferred embodiment of the binding agent used herein the second heavy chain comprises or consists essentially of or consists of an amino acid sequence set forth in SEQ ID NO: 24 or 30 [IgG1-Fc_FEAL], then in a further preferred embodiment of the binding agent used herein the first heavy chain comprises or consists essentially of or consists of an amino acid sequence set forth in SEQ ID NO: 23 or 29 [IgG1-Fc_FEAR] and the second heavy chain comprises or consists essentially of or consists of an amino acid sequence set forth in SEQ ID NO: 24 or 30 [IgG1-Fc_FEAL].
Preferably, the terms used herein are defined as described in “A multilingual glossary of biotechnological terms: (IUPAC Recommendations)”, H. G. W. Leuenberger, B. Nagel, and H. Kölbl, Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).
The practice of the present disclosure will employ, unless otherwise indicated, conventional chemistry, biochemistry, cell biology, immunology, and recombinant DNA techniques which are explained in the literature in the field (cf., e.g., Organikum, Deutscher Verlag der Wissenschaften, Berlin 1990; Streitwieser/Heathcook, “Organische Chemie”, VCH, 1990; Beyer/Walter, “Lehrbuch der Organischen Chemie”, S. Hirzel Verlag Stuttgart, 1988; Carey/Sundberg, “Organische Chemie”, VCH, 1995; March, “Advanced Organic Chemistry”, John Wiley & Sons, 1985; Römpp Chemie Lexikon, Falbe/Regitz (Hrsg.), Georg Thieme Verlag Stuttgart, New York, 1989; Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989.
All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by the context. The use of any and all examples, or exemplary language (e.g., “such as”), provided herein is intended merely to better illustrate the present disclosure and does not pose a limitation on the scope of the present disclosure otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the present disclosure.
Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein.
Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.
In the following, definitions will be provided which apply to all aspects of the present disclosure. The following terms have the following meanings unless otherwise indicated. Any undefined terms have their art recognized meanings.
Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated member, integer or step or group of members, integers or steps but not the exclusion of any other member, integer or step or group of members, integers or steps. The term “consisting essentially of” means excluding other members, integers or steps of any essential significance. The term “comprising” encompasses the term “consisting essentially of” which, in turn, encompasses the term “consisting of”. Thus, at each occurrence in the present application, the term “comprising” may be replaced with the term “consisting essentially of” or “consisting of”. Likewise, at each occurrence in the present application, the term “consisting essentially of” may be replaced with the term “consisting of”.
The terms “a”, “an” and “the” and similar references used in the context of describing the present disclosure (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by the context.
Where used herein, “and/or” is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example, “X and/or Y” is to be taken as specific disclosure of each of (i) X, (ii) Y, and (iii) X and Y, just as if each is set out individually herein.
In the context of the present disclosure, the term “about” denotes an interval of accuracy that the person of ordinary skill will understand to still ensure the technical effect of the feature in question. The term typically indicates deviation from the indicated numerical value by ±5%, ±4%, ±3%, ±2%, ±1%, ±0.9%, ±0.8%, ±0.7%, ±0.6%, ±0.5%, ±0.4%, ±0.3%, ±0.2%, ±0.1%, ±0.05%, and for example ±0.01%. As will be appreciated by the person of ordinary skill, the specific such deviation for a numerical value for a given technical effect will depend on the nature of the technical effect. For example, a natural or biological technical effect may generally have a larger such deviation than one for a man-made or engineering technical effect.
The term “binding agent” in the context of the present disclosure refers to any agent capable of binding to desired antigens. In certain embodiments of the present disclosure, the binding agent is an antibody, antibody fragment, or construct thereof. The binding agent may also comprise synthetic, modified or non-naturally occurring moieties, in particular non-peptide moieties. Such moieties may, for example, link desired antigen-binding functionalities or regions such as antibodies or antibody fragments. In one embodiment, the binding agent is a synthetic construct comprising antigen-binding CDRs or variable regions.
As used herein, “immune checkpoint” refers to regulators of the immune system, and, in particular, co-stimulatory and inhibitory signals that regulate the amplitude and quality of T cell receptor recognition of an antigen. In certain embodiments, the immune checkpoint is an inhibitory signal. In certain embodiments, the inhibitory signal is the interaction between PD-1 and PD-L1 and/or PD-L2. In certain embodiments, the inhibitory signal is the interaction between CTLA-4 and CD80 or CD86 to displace CD28 binding. In certain embodiments the inhibitory signal is the interaction between LAG-3 and MHC class II molecules. In certain embodiments, the inhibitory signal is the interaction between TIM-3 and one or more of its ligands, such as galectin 9, PtdSer, HMGB1 and CEACAM1. In certain embodiments, the inhibitory signal is the interaction between one or several KIRs and their ligands. In certain embodiments, the inhibitory signal is the interaction between TIGIT and one or more of its ligands, PVR, PVRL2 and PVRL3. In certain embodiments, the inhibitory signal is the interaction between CD94/NKG2A and HLA-E. In certain embodiments, the inhibitory signal is the interaction between VISTA and its binding partner(s). In certain embodiments, the inhibitory signal is the interaction between one or more Siglecs and their ligands. In certain embodiments, the inhibitory signal is the interaction between GARP and one or more of its ligands. In certain embodiments, the inhibitory signal is the interaction between CD47 and SIRPα. In certain embodiments, the inhibitory signal is the interaction between PVRIG and PVRL2. In certain embodiments, the inhibitory signal is the interaction between CSF1R and CSF1. In certain embodiments, the inhibitory signal is the interaction between BTLA and HVEM. In certain embodiments, the inhibitory signal is part of the adenosinergic pathway, e.g., the interaction between A2AR and/or A2BR and adenosine, produced by CD39 and CD73. In certain embodiments, the inhibitory signal is the interaction between B7-H3 and its receptor and/or B7-H4 and its receptor. In certain embodiments, the inhibitory signal is mediated by IDO, CD20, NOX or TDO.
The terms “checkpoint inhibitor” (CPI) and “immune checkpoint (ICP) inhibitor” are used herein synonymously. The terms refer to molecules, such as binding agents, which totally or partially reduce, inhibit, interfere with or negatively modulate one or more checkpoint proteins or that totally or partially reduce, inhibit, interfere with or negatively modulate expression of one or more checkpoint proteins, like molecules, such as binding agents, which inhibit an immune checkpoint, in particular, which inhibit the inhibitory signal of an immune checkpoint. In one embodiment, the immune checkpoint inhibitor binds to one or more checkpoint proteins. In one embodiment, the immune checkpoint inhibitor binds to one or more molecules regulating checkpoint proteins. In one embodiment, the immune checkpoint inhibitor binds to precursors of one or more checkpoint proteins e.g., on DNA- or RNA-level. Any agent that functions as a checkpoint inhibitor according to the present disclosure can be used. The term “partially” as used herein means at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% in the level, e.g., in the level of inhibition of a checkpoint protein.
In one embodiment, the checkpoint inhibitor can be any compound, such as any binding agent, which inhibits the inhibitory signal of an immune checkpoint, wherein the inhibitory signal is selected from the group consisting of: the interaction between PD-1 and PD-L1 and/or PD-L2; the interaction between CTLA-4 and CD80 or CD86 to displace CD28 binding; the interaction between LAG-3 and MHC class II molecules; the interaction between TIM-3 and one or more of its ligands, such as galectin 9, PtdSer, HMGB1 and CEACAM1; the interaction between one or several KIRs and their ligands; the interaction between TIGIT and one or more of its ligands, PVR, PVRL2 and PVRL3; the interaction between CD94/NKG2A and HLA-E; the interaction between VISTA and its binding partner(s); the interaction between one or more Siglecs and their ligands; the interaction between GARP and one or more of its ligands; the interaction between CD47 and SIRPα; the interaction between PVRIG and PVRL2; the interaction between CSF1R and CSF1; the interaction between BTLA and HVEM; part of the adenosinergic pathway, e.g., the interaction between A2AR and/or A2BR and adenosine, produced by CD39 and CD73; the interaction between B7-H3 and its receptor and/or B7-H4 and its receptor; an inhibitory signal mediated by IDO, CD20, NOX or TDO. In one embodiment, the checkpoint inhibitor is at least one selected from the group consisting of PD-1 inhibitors, PD-L1 inhibitors, PD-L2 inhibitors, CTLA-4 inhibitors, TIM-3 inhibitors, KIR inhibitors, LAG-3 inhibitors, TIGIT inhibitors, VISTA inhibitors, and GARP inhibitors. In one embodiment, the checkpoint inhibitor may be a blocking antibody, such as a PD-1 blocking antibody, a CTLA4 blocking antibody, a PD-L1 blocking antibody, a PD-L2 blocking antibody, a TIM-3 blocking antibody, a KIR blocking antibody, a LAG-3 blocking antibody, a TIGIT blocking antibody, a VISTA blocking antibody, or a GARP blocking antibody. Examples of a PD-1 blocking antibody include pembrolizumab, nivolumab, cemiplimab, and spartalizumab. Examples of a CTLA4 blocking antibody include ipilimumab and tremelimumab. Examples of a PD-L1 blocking antibody include atezolizumab, durvalumab, and avelumab.
The term “immunoglobulin” relates to proteins of the immunoglobulin superfamily, preferably to antigen receptors such as antibodies or the B cell receptor (BCR). The immunoglobulins are characterized by a structural domain, i.e., the immunoglobulin domain, having a characteristic immunoglobulin (Ig) fold. The term encompasses membrane bound immunoglobulins as well as soluble immunoglobulins. Membrane bound immunoglobulins are also termed surface immunoglobulins or membrane immunoglobulins, which are generally part of the BCR. Soluble immunoglobulins are generally termed antibodies.
The structure of immunoglobulins has been well characterized. See, e.g., Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)). Briefly, immunoglobulins generally comprise several chains, typically two identical heavy chains and two identical light chains which are linked via disulfide bonds. These chains are primarily composed of immunoglobulin domains or regions, such as the VL or VL (variable light chain) domain/region, CL or CL (constant light chain) domain/region, VH or VH (variable heavy chain) domain/region, and the CH or CH (constant heavy chain) domains/regions CH1 (CH1), CH2 (CH2), CH3 (CH3), and CH4 (CH4). The heavy chain constant region typically is comprised of three domains, CH1, CH2, and CH3. The hinge region is the region between the CH1 and CH2 domains of the heavy chain and is highly flexible. Disulfide bonds in the hinge region are part of the interactions between two heavy chains in an IgG molecule. Each light chain typically is comprised of a VL and a CL. The light chain constant region typically is comprised of one domain, CL. The VH and VL regions may be further subdivided into regions of hypervariability (or hypervariable regions which may be hypervariable in sequence and/or form of structurally defined loops), also termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk J. Mol. Biol. 196, 901-917 (1987)). Unless otherwise stated or contradicted by context, CDR sequences herein are identified according to IMGT rules using DomainGapAlign (Lefranc M P., Nucleic Acids Research 1999; 27:209-212 and Ehrenmann F., Kaas Q. and Lefranc M.-P. Nucleic Acids Res., 38, D301-307 (2010); see also internet http address www.imgt.org. Unless otherwise stated or contradicted by context, reference to amino acid positions in the constant regions in the present disclosure is according to the EU-numbering (Edelman et al., Proc Natl Acad Sci USA. 1969 May; 63(1):78-85; Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition. 1991 NIH Publication No. 91-3242).
There are five types of mammalian immunoglobulin heavy chains, i.e., α, δ, ε, γ, and μ which account for the different classes of antibodies, i.e., IgA, IgD, IgE, IgG, and IgM. As opposed to the heavy chains of soluble immunoglobulins, the heavy chains of membrane or surface immunoglobulins comprise a transmembrane domain and a short cytoplasmic domain at their carboxy-terminus. In mammals there are two types of light chains, i.e., lambda and kappa. The immunoglobulin chains comprise a variable region and a constant region. The constant region is essentially conserved within the different isotypes of the immunoglobulins, wherein the variable part is highly divers and accounts for antigen recognition.
The term “amino acid” and “amino acid residue” may herein be used interchangeably, and are not to be understood limiting. Amino acids are organic compounds containing amine (—NH2) and carboxyl (—COOH) functional groups, along with a side chain (R group) specific to each amino acid. In the context of the present disclosure, amino acids may be classified based on structure and chemical characteristics. Thus, classes of amino acids may be reflected in one or both of the following tables:
For the purposes of the present disclosure, “variants” of an amino acid sequence (peptide, protein or polypeptide) comprise amino acid insertion variants, amino acid addition variants, amino acid deletion variants and/or amino acid substitution variants. The term “variant” includes all mutants, splice variants, posttranslationally modified variants, conformations, isoforms, allelic variants, species variants, and species homologs, in particular those which are naturally occurring. The term “variant” includes, in particular, fragments of an amino acid sequence.
Amino acid insertion variants comprise insertions of single or two or more amino acids in a particular amino acid sequence. In the case of amino acid sequence variants having an insertion, one or more amino acid residues are inserted into a particular site in an amino acid sequence, although random insertion with appropriate screening of the resulting product is also possible.
Amino acid addition variants comprise amino- and/or carboxy-terminal fusions of one or more amino acids, such as 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids.
Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence, such as by removal of 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. The deletions may be in any position of the protein. Amino acid deletion variants that comprise the deletion at the N-terminal and/or C-terminal end of the protein are also called N-terminal and/or C-terminal truncation variants.
Amino acid substitution variants are characterized by at least one residue in the sequence being removed and another residue being inserted in its place. Substitution of one amino acid for another may be classified as a conservative or non-conservative substitution. Preference is given to the modifications being in positions in the amino acid sequence which are not conserved between homologous proteins or peptides and/or to replacing amino acids with other ones having similar properties. Preferably, amino acid changes in peptide and protein variants are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. A conservative amino acid change involves substitution of one of a family of amino acids which are related in their side chains. In the context of the present disclosure, a “conservative substitution” is a substitution of one amino acid with another amino acid having similar structural and/or chemical characteristics, such substitution of one amino acid residue for another amino acid residue of the same class as defined in any of the two tables above: for example, leucine may be substituted with isoleucine as they are both aliphatic, branched hydrophobes. Similarly, aspartic acid may be substituted with glutamic acid since they are both small, negatively charged residues. Naturally occurring amino acids may also be generally divided into four families: acidic (aspartate, glutamate), basic (lysine, arginine, histidine), non-polar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids. In one embodiment, conservative amino acid substitutions include substitutions within the following groups:
The term “amino acid corresponding to position . . . ” and similar expressions as used herein refer to an amino acid position number in a human IgG1 heavy chain. Corresponding amino acid positions in other immunoglobulins may be found by alignment with human IgG1. Thus, an amino acid or segment in one sequence that “corresponds to” an amino acid or segment in another sequence is one that aligns with the other amino acid or segment using a standard sequence alignment program such as ALIGN, ClustalW or similar, typically at default settings and has at least 50%, at least 80%, at least 90%, or at least 95% identity to a human IgG1 heavy chain. It is considered well-known in the art how to align a sequence or segment in a sequence and thereby determine the corresponding position in a sequence to an amino acid position according to the present disclosure.
The term “antibody” (Ab) in the context of the present disclosure refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative of either thereof, which has the ability to specifically bind to an antigen (in particular an epitope on an antigen) under typical physiological conditions, preferably with a half-life of significant periods of time, such as at least about 30 minutes, at least about 45 minutes, at least about one hour, at least about two hours, at least about four hours, at least about 8 hours, at least about 12 hours, about 24 hours or more, about 48 hours or more, about 3, 4, 5, 6, 7 or more days, etc., or any other relevant functionally-defined period (such as a time sufficient to induce, promote, enhance, and/or modulate a physiological response associated with antibody binding to the antigen and/or time sufficient for the antibody to recruit an effector activity). In particular, the term “antibody” refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. The term “antibody” includes monoclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, chimeric antibodies and combinations of any of the foregoing. Each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region (CH). Each light chain is comprised of a light chain variable region (VL) and a light chain constant region (CL). The variable regions and constant regions are also referred to herein as variable domains and constant domains, respectively. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The CDRs of a VH are termed HCDR1, HCDR2 and HCDR3 (or CDR-H1, CDR-H2 and CDR-H3), the CDRs of a VL are termed LCDR1, LCDR2 and LCDR3 (or CDR-L1, CDR-L2 and CDR-L3). The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of an antibody comprise the heavy chain constant region (CH) and the light chain constant region (CL), wherein CH can be further subdivided into constant domain CH1, a hinge region, and constant domains CH2 and CH3 (arranged from amino-terminus to carboxy-terminus in the following order: CH1, CH2, CH3). The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and components of the complement system such as C1q. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoactive portions of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. Antibodies may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab)2, as well as single chain antibodies and humanized antibodies.
The variable regions of the heavy and light chains of the immunoglobulin molecule contain a binding domain that interacts with an antigen. The terms “binding region” and “antigen-binding region” are used herein interchangeably and refer to the region which interacts with the antigen and comprises both a VH region and a VL region. An antibody as used herein comprises not only monospecific antibodies, but also multispecific antibodies which comprise multiple, such as two or more, e.g., three or more, different antigen-binding regions.
As indicated above, the term antibody herein, unless otherwise stated or clearly contradicted by context, includes fragments of an antibody that are antigen-binding fragments, i.e., retain the ability to specifically bind to the antigen. It has been shown that the antigen-binding function of an antibody may be performed by fragments of a full-length antibody. Examples of antigen-binding fragments encompassed within the term “antibody” include (i) a Fab′ or Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains, or a monovalent antibody as described in WO 2007/059782 (Genmab); (ii) F(ab′)2 fragments, bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting essentially of the VH and CH1 domains; (iv) a Fv fragment consisting essentially of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment (Ward et al., Nature 341, 544-546 (1989)), which consists essentially of a VH domain and also called domain antibodies (Holt et al; Trends Biotechnol. 2003 November; 21(11):484-90); (vi) camelid or Nanobody molecules (Revets et al; Expert Opin Biol Ther. 2005 January; 1(1):111-24); and (vii) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they may be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain antibodies or single chain Fv (scFv), see for instance Bird et al., Science 242, 423-426 (1988) and Huston et al., PNAS USA 85, 5879-5883 (1988)). Such single chain antibodies are encompassed within the term antibody unless otherwise noted or clearly indicated by context. Although such fragments are generally included within the meaning of antibody, they collectively and each independently are unique features of the present disclosure, exhibiting different biological properties and utility. These and other useful antibody fragments in the context of the present disclosure, as well as bispecific formats of such fragments, are discussed further herein. It also should be understood that the term antibody, unless specified otherwise, also includes polyclonal antibodies, monoclonal antibodies (mAbs), antibody-like polypeptides, such as chimeric antibodies and humanized antibodies, and antibody fragments retaining the ability to specifically bind to the antigen (antigen-binding fragments) provided by any known technique, such as enzymatic cleavage, peptide synthesis, and recombinant techniques.
An antibody as generated can possess any isotype. As used herein, the term “isotype” refers to the immunoglobulin class (for instance IgG (such as IgG1, IgG2, IgG3, IgG4), IgD, IgA (such as IgA1, IgA2), IgE, IgM, or IgY) that is encoded by heavy chain constant region genes. When a particular isotype, e.g. IgG1, is mentioned herein, the term is not limited to a specific isotype sequence, e.g. a particular IgG1 sequence, but is used to indicate that the antibody is closer in sequence to that isotype, e.g. IgG1, than to other isotypes. Thus, e.g. an IgG1 antibody disclosed herein may be a sequence variant of a naturally-occurring IgG1 antibody, including variations in the constant regions.
IgG1 antibodies can exist in multiple polymorphic variants termed allotypes (reviewed in Jefferis and Lefranc 2009. mAbs Vol 1 Issue 4 1-7) any of which are suitable for use in some of the embodiments herein. Common allotypic variants in human populations are those designated by the letters a, f, n, z or combinations thereof. In any of the embodiments herein, the antibody may comprise a heavy chain Fc region comprising a human IgG Fc region. In further embodiments, the human IgG Fc region comprises a human IgG1.
The term “multispecific antibody” in the context of the present disclosure refers to an antibody having at least two different antigen-binding regions defined by different antibody sequences. In some embodiments, said different antigen-binding regions bind different epitopes on the same antigen. However, in preferred embodiments, said different antigen-binding regions bind different target antigens. In one embodiment, the multispecific antibody is a “bispecific antibody” or “bs”. A multispecific antibody, such as a bispecific antibody, can be of any format, including any of the bispecific or multispecific antibody formats described herein below.
The term “full-length” when used in the context of an antibody indicates that the antibody is not a fragment, but contains all of the domains of the particular isotype normally found for that isotype in nature, e.g. the VH, CH1, CH2, CH3, hinge, VL and CL domains for an IgG1 antibody.
The term “human antibody”, as used herein, is intended to include antibodies having variable and framework regions derived from human germline immunoglobulin sequences and a human immunoglobulin constant domain. The human antibodies disclosed herein may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations, insertions or deletions introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term “human antibody”, as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another non-human species, such as a mouse, have been grafted onto human framework sequences.
The term “chimeric antibody” as used herein, refers to an antibody wherein the variable region is derived from a non-human species (e.g. derived from rodents) and the constant region is derived from a different species, such as human. Chimeric antibodies may be generated by antibody engineering. “Antibody engineering” is a term used generically for different kinds of modifications of antibodies, and processes for antibody engineering are well-known for the skilled person. In particular, a chimeric antibody may be generated by using standard DNA techniques as described in Sambrook et al., 1989, Molecular Cloning: A laboratory Manual, New York: Cold Spring Harbor Laboratory Press, Ch. 15. Thus, the chimeric antibody may be a genetically or an enzymatically engineered recombinant antibody. It is within the knowledge of the skilled person to generate a chimeric antibody, and thus, generation of the chimeric antibody may be performed by other methods than those described herein. Chimeric monoclonal antibodies for therapeutic applications in humans are developed to reduce anticipated antibody immunogenicity of non-human antibodies, e.g. rodent antibodies. They may typically contain non-human (e.g. murine or rabbit) variable regions, which are specific for the antigen of interest, and human constant antibody heavy and light chain domains. The terms “variable region” or “variable domain” as used in the context of chimeric antibodies, refer to a region which comprises the CDRs and framework regions of both the heavy and light chains of an immunoglobulin, as described below.
The term “humanized antibody” as used herein, refers to a genetically engineered non-human antibody, which contains human antibody constant domains and non-human variable domains modified to contain a high level of sequence homology to human variable domains. This can be achieved by grafting of the six non-human antibody complementarity-determining regions (CDRs), which together form the antigen binding site, onto a homologous human acceptor framework region (FR) (see WO 92/22653 and EP 0 629 240). In order to fully reconstitute the binding affinity and specificity of the parental antibody, the substitution of framework residues from the parental antibody (i.e. the non-human antibody) into the human framework regions (back-mutations) may be required. Structural homology modeling may help to identify the amino acid residues in the framework regions that are important for the binding properties of the antibody. Thus, a humanized antibody may comprise non-human CDR sequences, primarily human framework regions optionally comprising one or more amino acid back-mutations to the non-human amino acid sequence, and fully human constant regions. Optionally, additional amino acid modifications, which are not necessarily back-mutations, may be applied to obtain a humanized antibody with preferred characteristics, such as affinity and biochemical properties.
As used herein, a protein which is “derived from” another protein, e.g., a parent protein, means that one or more amino acid sequences of the protein are identical or similar to one or more amino acid sequences in the other or parent protein. For example, in an antibody, binding arm, antigen-binding region, constant region, or the like which is derived from another or a parent antibody, binding arm, antigen-binding region, or constant region, one or more amino acid sequences are identical or similar to those of the other or parent antibody, binding arm, antigen-binding region, or constant region. Examples of such one or more amino acid sequences include, but are not limited to, those of the VH and VL CDRs and/or one or more or all of the framework regions, VH, VL, CL, hinge, or CH regions. For example, a humanized antibody can be described herein as “derived from” a non-human parent antibody, meaning that at least the VL and VH CDR sequences are identical or similar to the VH and VL CDR sequences of said non-human parent antibody. A chimeric antibody can be described herein as being “derived from” a non-human parent antibody, meaning that typically the VH and VL sequences may be identical or similar to those of the non-human parent antibody. Another example is a binding arm or an antigen-binding region which may be described herein as being “derived from” a particular parent antibody, meaning that said binding arm or antigen-binding region typically comprises identical or similar VH and/or VL CDRs, or VH and/or VL sequences to the binding arm or antigen-binding region of said parent antibody. As described elsewhere herein, however, amino acid modifications such as mutations can be made in the CDRs, constant regions or elsewhere in the antibody, binding arm, antigen-binding region or the like, to introduce desired characteristics. When used in the context of one or more sequences derived from a first or parent protein, a “similar” amino acid sequence preferably has a sequence identity of at least about 50%, such as at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 97%, 98% or 99%.
Non-human antibodies can be generated in a number of different species, such as mouse, rabbit, chicken, guinea pig, llama and goat.
Monoclonal antibodies can be produced by a variety of techniques, including conventional monoclonal antibody methodology, e.g., the standard somatic cell hybridization technique of Kohler and Milstein, Nature 256: 495 (1975). Other techniques for producing monoclonal antibodies can be employed, e.g., viral or oncogenic transformation of B-lymphocytes or phage display techniques using libraries of antibody genes, and such methods are well known to a person skilled in the art.
Hybridoma production in such non-human species is a very well-established procedure. Immunization protocols and techniques for isolation of splenocytes of immunized animals/non-human species for fusion are known in the art. Fusion partners (e.g., murine myeloma cells) and fusion procedures are also known.
When used herein, unless contradicted by context, the term “Fab-arm” or “arm” refers to one heavy chain-light chain pair and is used interchangeably with “half molecules” herein.
The term “binding arm comprising an antigen-binding region” means an antibody molecule or fragment that comprises an antigen-binding region. Thus, a binding arm can comprise, e.g., the six VH and VL CDR sequences, the VH and VL sequences, a Fab or Fab′ fragment, or a Fab-arm.
When used herein, unless contradicted by context, the term “Fc region” refers to an antibody region consisting of the two Fc sequences of the heavy chains of an immunoglobulin, wherein said Fc sequences comprise at least a hinge region, a CH2 domain, and a CH3 domain. In one embodiment, the term “Fc region”, as used herein, refers to a region comprising, in the direction from the N- to C-terminal end of the antibody, at least a hinge region, a CH2 region and a CH3 region. An Fc region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells) and components of the complement system.
In the context of the present disclosure, the term “induce Fc-mediated effector function to a lesser extent” used in relation to an antibody, including a multispecific antibody, means that the antibody induces Fc-mediated effector functions, such function in particular being selected from the list of IgG Fc receptor (FcgammaR, FcγR) binding, C1q binding, ADCC or CDC, to a lesser extent compared to a human IgG1 antibody comprising (i) the same CDR sequences, in particular comprising the same first and second antigen-binding regions, as said antibody and (ii) two heavy chains comprising human IgG1 hinge, CH2 and CH3 regions.
Fc-mediated effector function may be measured by binding to FcγRs, binding to C1q, or induction of Fc-mediated cross-linking via FcγRs.
The term “hinge region” as used herein refers to the hinge region of an immunoglobulin heavy chain. Thus, for example, the hinge region of a human IgG1 antibody corresponds to amino acids 216-230 according to the EU numbering as set forth in Kabat (Kabat, E. A. et al., Sequences of proteins of immunological interest. 5th Edition—US Department of Health and Human Services, NIH publication No. 91-3242, pp 662,680,689 (1991). However, the hinge region may also be any of the other subtypes as described herein.
The term “CH1 region” or “CH1 domain” as used herein refers to the CH1 region of an immunoglobulin heavy chain. Thus, for example, the CH1 region of a human IgG1 antibody corresponds to amino acids 118-215 according to the EU numbering as set forth in Kabat (ibid). However, the CH1 region may also be any of the other subtypes as described herein.
The term “CH2 region” or “CH2 domain” as used herein refers to the CH2 region of an immunoglobulin heavy chain. Thus, for example, the CH2 region of a human IgG1 antibody corresponds to amino acids 231-340 according to the EU numbering as set forth in Kabat (ibid). However, the CH2 region may also be any of the other subtypes as described herein.
The term “CH3 region” or “CH3 domain” as used herein refers to the CH3 region of an immunoglobulin heavy chain. Thus, for example, the CH3 region of a human IgG1 antibody corresponds to amino acids 341-447 according to the EU numbering as set forth in Kabat (ibid). However, the CH3 region may also be any of the other subtypes as described herein.
The term “monovalent antibody” means in the context of the present disclosure that an antibody molecule is capable of binding a single molecule of the antigen, and thus is not capable of antigen cross-linking.
A “CD137 antibody” or “anti-CD137 antibody” is an antibody as described above, which binds specifically to the antigen CD137.
A “CD137×PD-L1 antibody” or “anti-CD137×PD-L1 antibody” is a bispecific antibody, which comprises two different antigen-binding regions, one of which binds specifically to the antigen CD137 and one of which binds specifically to the antigen PD-L1.
The term “biosimilar” (e.g., of an approved reference product/biological drug) as used herein refers to a biologic product that is similar to the reference product based on data from (a) analytical studies demonstrating that the biological product is highly similar to the reference product notwithstanding minor differences in clinically inactive components; (b) animal studies (including the assessment of toxicity); and/or (c) a clinical study or studies (including the assessment of immunogenicity and pharmacokinetics or pharmacodynamics) that are sufficient to demonstrate safety, purity, and potency in one or more appropriate conditions of use for which the reference product is approved and intended to be used and for which approval is sought (e.g., that there are no clinically meaningful differences between the biological product and the reference product in terms of the safety, purity, and potency of the product). In some embodiments, the biosimilar biological product and reference product utilizes the same mechanism or mechanisms of action for the condition or conditions of use prescribed, recommended, or suggested in the proposed labeling, but only to the extent the mechanism or mechanisms of action are known for the reference product. In some embodiments, the condition or conditions of use prescribed, recommended, or suggested in the labeling proposed for the biological product have been previously approved for the reference product. In some embodiments, the route of administration, the dosage form, and/or the strength of the biological product are the same as those of the reference product. A biosimilar can be, e.g., a presently known antibody having the same primary amino acid sequence as a marketed antibody, but may be made in different cell types or by different production, purification, or formulation methods.
As used herein, the terms “binding” or “capable of binding” in the context of the binding of an antibody to a predetermined antigen or epitope typically is a binding with an affinity corresponding to a KD of about 10−7 M or less, such as about 10−8 M or less, such as about 10−9 M or less, about 10−10 M or less, or about 10−7 M or even less, when determined using Bio-Layer Interferometry (BLI) or, for instance, when determined using surface plasmon resonance (SPR) technology in a BIAcore 3000 instrument using the antigen as the ligand and the antibody as the analyte. The antibody binds to the predetermined antigen with an affinity corresponding to a KD that is at least ten-fold lower, such as at least 100-fold lower, for instance at least 1,000-fold lower, such as at least 10,000-fold lower, for instance at least 100,000-fold lower than its KD for binding to a non-specific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely related antigen. The amount with which the affinity is higher is dependent on the KD of the antibody, so that when the KD of the antibody is very low (that is, the antibody is highly specific), then the degree to which the affinity for the antigen is lower than the affinity for a non-specific antigen may be at least 10,000-fold.
The term “kd” (sec−1), as used herein, refers to the dissociation rate constant of a particular antibody-antigen interaction. Said value is also referred to as the koff value.
The term “KD” (M), as used herein, refers to the dissociation equilibrium constant of a particular antibody-antigen interaction.
Two antibodies have the “same specificity” if they bind to the same antigen and to the same epitope. Whether an antibody to be tested recognizes the same epitope as a certain antigen-binding antibody, i.e., the antibodies bind to the same epitope, may be tested by different methods well known to a person skilled in the art.
The competition between the antibodies can be detected by a cross-blocking assay. For example, a competitive ELISA assay may be used as a cross-blocking assay. E.g., target antigen may be coated on the wells of a microtiter plate and antigen-binding antibody and candidate competing test antibody may be added. The amount of the antigen-binding antibody bound to the antigen in the well indirectly correlates with the binding ability of the candidate competing test antibody that competes therewith for binding to the same epitope. Specifically, the larger the affinity of the candidate competing test antibody is for the same epitope, the smaller the amount of the antigen-binding antibody bound to the antigen-coated well. The amount of the antigen-binding antibody bound to the well can be measured by labeling the antibody with detectable or measurable labeling substances.
An antibody competing for binding to an antigen with another antibody, e.g., an antibody comprising heavy and light chain variable regions as described herein, or an antibody having the specificity for an antigen of another antibody, e.g., an antibody comprising heavy and light chain variable regions as described herein, may be an antibody comprising variants of said heavy and/or light chain variable regions as described herein, e.g. modifications in the CDRs and/or a certain degree of identity as described herein.
An “isolated multispecific antibody” as used herein is intended to refer to a multispecific antibody which is substantially free of other antibodies having different antigenic specificities (for instance an isolated bispecific antibody that specifically binds to CD137 and PD-L1 is substantially free of monospecific antibodies that specifically bind to CD137 or PD-L1).
The term “monoclonal antibody” as used herein refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.
When used herein the term “heterodimeric interaction between the first and second CH3 regions” refers to the interaction between the first CH3 region and the second CH3 region in a first-CH3/second-CH3 heterodimeric antibody.
When used herein the term “homodimeric interactions of the first and second CH3 regions” refers to the interaction between a first CH3 region and another first CH3 region in a first-CH3/first-CH3 homodimeric antibody and the interaction between a second CH3 region and another second CH3 region in a second-CH3/second-CH3 homodimeric antibody.
When used herein the term “homodimeric antibody” refers to an antibody comprising two first Fab-arms or half-molecules, wherein the amino acid sequence of said Fab-arms or half-molecules is the same.
When used herein the term “heterodimeric antibody” refers to an antibody comprising a first and a second Fab-arm or half-molecule, wherein the amino acid sequence of said first and second Fab-arms or half-molecules are different. In particular, the CH3 region, or the antigen-binding region, or the CH3 region and the antigen-binding region of said first and second Fab-arms/half-molecules are different.
The term “reducing conditions” or “reducing environment” refers to a condition or an environment in which a substrate, such as a cysteine residue in the hinge region of an antibody, is more likely to become reduced than oxidized.
The present disclosure also describes multispecific antibodies, such as bispecific antibodies, comprising functional variants of the VL regions, VH regions, or one or more CDRs of the bispecific antibodies of the examples. A functional variant of a VL, VH, or CDR used in the context of a bispecific antibody still allows each antigen-binding region of the bispecific antibody to retain at least a substantial proportion (at least about 50%, 60%, 70%, 80%, 90%, 95% or more) of the affinity and/or the specificity/selectivity of the parent bispecific antibody and in some cases such a bispecific antibody may be associated with greater affinity, selectivity and/or specificity than the parent bispecific antibody.
Such functional variants typically retain significant sequence identity to the parent bispecific antibody. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology=# of identical positions/total # of positions×100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The percent identity between two nucleotide or amino acid sequences may e.g. be determined using the algorithm of E. Meyers and W. Miller, Comput. Appl. Biosci 4, 11-17 (1988) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent identity between two amino acid sequences may be determined using the Needleman and Wunsch, J. Mol. Biol. 48, 444-453 (1970) algorithm.
In the context of the present disclosure, unless otherwise indicated, the following notations are used to describe a mutation: i) substitution of an amino acid in a given position is written as e.g. K409R which means a substitution of a lysine in position 409 of the protein with an arginine; and ii) for specific variants the specific three or one letter codes are used, including the codes Xaa and X to indicate any amino acid residue. Thus, the substitution of lysine with arginine in position 409 is designated as: K409R, and the substitution of lysine with any amino acid residue in position 409 is designated as K409X. In case of deletion of lysine in position 409 it is indicated by K409*.
Exemplary variants include those which differ from the VH and/or VL and/or CDRs of the parent sequences mainly by conservative substitutions; for example, 12, such as 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 of the substitutions in the variant are conservative amino acid residue replacements.
In the context of the present disclosure, conservative substitutions may be defined by substitutions within the classes of amino acids as defined in tables 2 and 3.
The term “CD137” as used herein, refers to CD137 (4-1BB), also referred to as tumor necrosis factor receptor superfamily member 9 (TNFRSF9), which is the receptor for the ligand TNFSF9/4-1BBL. CD137 (4-1BB) is believed to be involved in T-cell activation. Other synonyms for CD137 include, but are not limited to, 4-1BB ligand receptor, CDw137, T-cell antigen 4-1BB homolog and T-cell antigen ILA. In one embodiment, CD137 (4-1BB) is human CD137 (4-1BB), having UniProt accession number Q07011. The sequence of human CD137 is also shown in SEQ ID NO: 37. Amino acids 1-23 of SEQ ID NO: 37 correspond to the signal peptide of human CD137; while amino acids 24-186 of SEQ ID NO: 37 correspond to the extracellular domain of human CD137; and the remainder of the protein, i.e. from amino acids 187-213 and 214-255 of SEQ ID NO: 37 are transmembrane and cytoplasmic domain, respectively.
The “Programmed Death-1 (PD-1)” receptor refers to an immuno-inhibitory receptor belonging to the CD28 family. PD-1 (also known as CD279) is expressed predominantly on previously activated T cells in vivo, and binds to two ligands, PD-L1 (also known as B7-H1 or CD274) and PD-L2 (also known as B7-DC or CD273). The term “PD-1” as used herein includes human PD-1 (hPD-1), variants, isoforms, and species homologs of hPD-1, and analogs having at least one common epitope with hPD-1. The sequence of human PD-1 is also shown in SEQ ID NO: 39. “Programmed Death Ligand-1 (PD-L1)” is one of two cell surface glycoprotein ligands for PD-1 (the other being PD-L2) that downregulates T cell activation and cytokine secretion upon binding to PD-1.
The term “PD-L1” as used herein includes human PD-L1 (hPD-L1), variants, isoforms, and species homologs of hPD-L1, such as macaque (cynomolgus monkey), African elephant, wild boar and mouse PD-L1 (cf., e.g., Genbank accession no. NP_054862.1, XP_005581836, XP_003413533, XP_005665023 and NP_068693, respectively), and analogs having at least one common epitope with hPD-L1. The sequence of human PD-L1 is also shown in SEQ ID NO: 40, wherein amino acids 1-18 are predicted to be a signal peptide. The term “PD-L2” as used herein includes human PD-L2 (hPD-L2), variants, isoforms, and species homologs of hPD-L2, and analogs having at least one common epitope with hPD-L2. The ligands of PD-1 (PD-L1 and PD-L2) are expressed on the surface of antigen-presenting cells, such as dendritic cells or macrophages, and other immune cells. Binding of PD-1 to PD-L1 or PD-L2 results in downregulation of T cell activation. Cancer cells expressing PD-L1 and/or PD-L2 are able to switch off T cells expressing PD-1 what results in suppression of the anticancer immune response. The interaction between PD-1 and its ligands results in a decrease in tumor infiltrating lymphocytes, a decrease in T cell receptor mediated proliferation, and immune evasion by the cancerous cells. Immune suppression can be reversed by inhibiting the local interaction of PD-1 with PD-L1, and the effect is additive when the interaction of PD-1 with PD-L2 is blocked as well.
The term “dysfunctional”, as used herein, refers to an immune cell that is in a state of reduced immune responsiveness to antigen stimulation. Dysfunctional includes unresponsive to antigen recognition and impaired capacity to translate antigen recognition into downstream T cell effector functions, such as proliferation, cytokine production (e.g., IL-2) and/or target cell killing.
The term “anergy”, as used herein, refers to the state of unresponsiveness to antigen stimulation resulting from incomplete or insufficient signals delivered through the T cell receptor (TCR). T cell anergy can also result upon stimulation with antigen in the absence of co-stimulation, resulting in the cell becoming refractory to subsequent activation by the antigen even in the context of co-stimulation. The unresponsive state can often be overridden by the presence of IL-2. Anergic T cells do not undergo clonal expansion and/or acquire effector functions.
The term “exhaustion”, as used herein, refers to immune cell exhaustion, such as T cell exhaustion as a state of T cell dysfunction that arises from sustained TCR signaling that occurs during many chronic infections and cancer. It is distinguished from anergy in that it arises not through incomplete or deficient signaling, but from sustained signaling. Exhaustion is defined by poor effector function, sustained expression of inhibitory receptors and a transcriptional state distinct from that of functional effector or memory T cells. Exhaustion prevents optimal control of diseases (e.g., infection and tumors). Exhaustion can result from both extrinsic negative regulatory pathways (e.g., immunoregulatory cytokines) as well as cell intrinsic negative regulatory pathways (inhibitory immune checkpoint pathways, such as described herein).
“Enhancing T cell function” means to induce, cause or stimulate a T cell to have a sustained or amplified biological function, or renew or reactivate exhausted or inactive T cells. Examples of enhancing T cell function include increased secretion of y-interferon from CD8+ T cells, increased proliferation, increased antigen responsiveness (e.g., tumor clearance) relative to such levels before the intervention. In one embodiment, the level of enhancement is as least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 200%, or more. Manners of measuring this enhancement are known to one of ordinary skill in the art.
The term “inhibitory nucleic acid” or “inhibitory nucleic acid molecule” as used herein refers to a nucleic acid molecule, e.g., DNA or RNA, that totally or partially reduces, inhibits, interferes with or negatively modulates one or more PD-1 proteins. Inhibitory nucleic acid molecules include, without limitation, oligonucleotides, siRNA, shRNA, antisense DNA or RNA molecules, and aptamers (e.g., DNA or RNA aptamers).
The term “oligonucleotide” as used herein refers to a nucleic acid molecule that is able to decrease protein expression, in particular expression of a PD-1 protein, such as the PD-1 proteins described herein. Oligonucleotides are short DNA or RNA molecules, typically comprising from 2 to 50 nucleotides. Oligonucleotides maybe single-stranded or double-stranded. A PD-1 inhibitor oligonucleotide may be an antisense-oligonucleotide.
Antisense-oligonucleotides are single-stranded DNA or RNA molecules that are complementary to a given sequence, in particular to a sequence of the nucleic acid sequence (or a fragment thereof) of a PD-1 protein. Antisense RNA is typically used to prevent protein translation of mRNA, e.g., of mRNA encoding a PD-1 protein, by binding to said mRNA. Antisense DNA is typically used to target a specific, complementary (coding or non-coding) RNA. If binding takes place, such a DNA/RNA hybrid can be degraded by the enzyme RNase H. Moreover, morpholino antisense oligonucleotides can be used for gene knockdowns in vertebrates. For example, Kryczek et al., 2006 (J Exp Med, 203:871-81) designed B7-H4-specific morpholinos that specifically blocked B7-H4 expression in macrophages, resulting in increased T cell proliferation and reduced tumor volumes in mice with tumor associated antigen (TAA)-specific T cells.
The terms “siRNA” or “small interfering RNA” or “small inhibitory RNA” are used interchangeably herein and refer to a double-stranded RNA molecule with a typical length of 20-25 base pairs that interferes with expression of a specific gene, such as a gene coding for a PD-1 protein, with a complementary nucleotide sequence. In one embodiment, siRNA interferes with mRNA therefore blocking translation, e.g., translation of a PD-1 protein. Transfection of exogenous siRNA may be used for gene knockdown, however, the effect maybe only transient, especially in rapidly dividing cells. Stable transfection may be achieved, e.g., by RNA modification or by using an expression vector. Useful modifications and vectors for stable transfection of cells with siRNA are known in the art. siRNA sequences may also be modified to introduce a short loop between the two strands resulting in a “small hairpin RNA” or “shRNA”. shRNA can be processed into a functional siRNA by Dicer. shRNA has a relatively low rate of degradation and turnover. Accordingly, the PD-1 inhibitor may be a shRNA.
The term “aptamer” as used herein refers to a single-stranded nucleic acid molecule, such as DNA or RNA, typically in a length of 25-70 nucleotides that is capable of binding to a target molecule, such as a polypeptide. In one embodiment, the aptamer binds to a PD-1 protein such as the PD-1 proteins described herein. For example, an aptamer according to the disclosure can specifically bind to a PD-1 protein or polypeptide, or to a molecule in a signaling pathway that modulates the expression of a PD-1 protein or polypeptide. The generation and therapeutic use of aptamers is well known in the art (see, e.g., U.S. Pat. No. 5,475,096).
The terms “small molecule inhibitor” or “small molecule” are used interchangeably herein and refer to a low molecular weight organic compound, usually up to 1000 daltons, that totally or partially reduces, inhibits, interferes with, or negatively modulates one or more PD-1 proteins as described above. Such small molecular inhibitors are usually synthesized by organic chemistry, but may also be isolated from natural sources, such as plants, fungi, and microbes. The small molecular weight allows a small molecule inhibitor to rapidly diffuse across cell membranes. For example, various A2AR antagonists known in the art are organic compounds having a molecular weight below 500 daltons.
The term “cell based therapy” refers to the transplantation of cells (e.g., T lymphocytes, dendritic cells, or stem cells) expressing a PD-1 inhibitor into a subject for the purpose of treating a disease or disorder (e.g., a cancer disease).
The term “oncolytic virus” as used herein, refers to a virus capable of selectively replicating in and slowing the growth or inducing the death of a cancerous or hyperproliferative cell, either in vitro or in vivo, while having no or minimal effect on normal cells. An oncolytic virus for the delivery of a PD-1 inhibitor comprises an expression cassette that may encode a PD-1 inhibitor that is an inhibitory nucleic acid molecule, such as a siRNA, shRNA, an oligonucleotide, antisense DNA or RNA, an aptamer, an antibody or a fragment thereof or a soluble PD-1 protein or fusion. The oncolytic virus preferably is replication competent and the expression cassette is under the control of a viral promoter, e.g., synthetic early/late poxvirus promoter. Exemplary oncolytic viruses include vesicular stomatitis virus (VSV), rhabdoviruses (e.g., picornaviruses such as Seneca Valley virus; SVV-001), coxsackievirus, parvovirus, Newcastle disease virus (NDV), herpes simplex virus (HSV; OncoVEX GMCSF), retroviruses (e.g., influenza viruses), measles virus, reovirus, Sinbis virus, vaccinia virus, as exemplarily described in WO 2017/209053 (including Copenhagen, Western Reserve, Wyeth strains), and adenovirus (e.g., Delta-24, Delta-24-RGD, ICOVIR-5, ICOVIR-7, Onyx-015, ColoAd1, H101, AD5/3-D24-GMCSF). Generation of recombinant oncolytic viruses comprising a soluble form of a PD-1 inhibitor and methods for their use are disclosed in WO 2018/022831, herein incorporated by reference in its entirety. Oncolytic viruses can be used as attenuated viruses.
“Treatment cycle” is herein defined as the time period, within the effects of separate dosages of the binding agent add on due to the pharmacodynamics of the binding agent, or in other words the time period after the subject's body is essentially cleared from the administrated biding agent. Multiple small doses in a small time window, e.g. within 2-24 few hours, such as 2-12 hours or on the same day, might be equal to a larger single dose.
In the present context, the term “treatment”, “treating” or “therapeutic intervention” relates to the management and care of a subject for the purpose of combating a condition such as a disease or disorder. The term is intended to include the full spectrum of treatments for a given condition from which the subject is suffering, such as administration of the therapeutically effective compound to alleviate the symptoms or complications, to delay the progression of the disease, disorder or condition, to alleviate or relief the symptoms and complications, and/or to cure or eliminate the disease, disorder or condition as well as to prevent the condition, wherein prevention is to be understood as the management and care of an individual for the purpose of combating the disease, condition or disorder and includes the administration of the active compounds to prevent the onset of the symptoms or complications. In one embodiment, “treatment” refers to the administration of an effective amount of a therapeutically active binding agent, such as of a therapeutically active antibody, of the present disclosure with the purpose of easing, ameliorating, arresting or eradicating (curing) symptoms or disease states.
The response to treatment as well as the resistance to, failure to respond to and/or relapse from treatment with a binding agent of the present disclosure may be determined according to the Response Evaluation Criteria in Solid Tumors; version 1.1 (RECIST Criteria v1.1). The RECIST Criteria are set forth in the table below (LD: longest dimension).
The “best overall response” is the best response recorded from the start of the treatment until disease progression/recurrence (the smallest measurements recorded since the treatment started will be used as the reference for PD). Subjects with CR or PR are considered to be objective response. Subjects with CR, PR or SD are considered to be in disease control. Subjects with NE are counted as non-responders. The best overall response is the best response recorded from the start of the treatment until disease progression/recurrence (the smallest measurements recorded since the treatment started will be used as the reference for PD). Subjects with CR, PR or SD are considered to be in disease control. Subjects with NE are counted as non-responders.
“Duration of response (DOR)” only applies to subjects whose confirmed best overall response is CR or PR and is defined as the time from the first documentation of objective tumor response (CR or PR) to the date of first PD or death due to underlying cancer.
“Progression-free survival (PFS)” is defined as the number of days from Day 1 in Cycle 1 to the first documented progression or death due to any cause.
“Overall survival (OS)” is defined as the number of days from Day 1 in Cycle 1 to death due to any cause. If a subject is not known to have died, then OS will be censored at the latest date the subject was known to be alive (on or before the cut-off date).
In the context of the present disclosure, the term “treatment regimen” refers to a structured treatment plan designed to improve and maintain health.
The term “effective amount” or “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result. A therapeutically effective amount of a binding agent, such as an antibody, like a multispecific antibody or monoclonal antibody, may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the binding agent to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the binding agent or a fragment thereof, are outweighed by the therapeutically beneficial effects. In the case that a reaction in a patient is insufficient with an initial dose, higher doses (or effectively higher doses achieved by a different, more localized route of administration) may be used. In case that unwanted side effects occur in a patient with a dose, lower doses (or effectively lower doses achieved by a different, more localized route of administration) may be used.
As used herein, the term “cancer” includes a disease characterized by aberrantly regulated cellular growth, proliferation, differentiation, adhesion, and/or migration. By “cancer cell” is meant an abnormal cell that grows by a rapid, uncontrolled cellular proliferation and continues to grow after the stimuli that initiated the new growth cease.
The term “cancer” according to the present disclosure also comprises cancer metastases. By “metastasis” is meant the spread of cancer cells from its original site to another part of the body. The formation of metastasis is a very complex process and depends on detachment of malignant cells from the primary tumor, invasion of the extracellular matrix, penetration of the endothelial basement membranes to enter the body cavity and vessels, and then, after being transported by the blood, infiltration of target organs. Finally, the growth of a new tumor, i.e. a secondary tumor or metastatic tumor, at the target site depends on angiogenesis. Tumor metastasis often occurs even after the removal of the primary tumor because tumor cells or components may remain and develop metastatic potential. In one embodiment, the term “metastasis” according to the present disclosure relates to “distant metastasis” which relates to a metastasis which is remote from the primary tumor and the regional lymph node system.
Terms such as “reduce”, “inhibit”, “interfere”, and “negatively modulate” as used herein means the ability to cause an overall decrease, for example, of about 5% or greater, about 10% or greater, about 15% or greater, about 20% or greater, about 25% or greater, about 30% or greater, about 40% or greater, about 50% or greater, or about 75% or greater, in the level. The term “inhibit” or similar phrases includes a complete or essentially complete inhibition, i.e. a reduction to zero or essentially to zero.
Terms such as “increase” or “enhance” in one embodiment relate to an increase or enhancement by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 80%, or at least about 100%.
“Physiological pH” as used herein refers to a pH of 7.5 or about 7.5.
As used in the present disclosure, “% by weight” refers to weight percent, which is a unit of concentration measuring the amount of a substance in grams (g) expressed as a percent of the total weight of the total composition in grams (g).
The term “TPS” or “tumor proportion score,” refers to the percentage of tumor cells expressing PD-L1 on the cell membrane. TPS typically includes the percentage of neoplastic cells expressing PD-L1 at any intensity (weak, moderate, or strong), which can be determined using an immunohistochemical assay using a diagnostic anti-human PD-L1 mAb, e.g. antibody 20C3 and antibody 22C3, described in WO 2014/100079. Cells are considered to express PD-L1 if membrane staining is present, including cells with partial membrane staining.
The term “freezing” relates to the solidification of a liquid, usually with the removal of heat.
The term “lyophilizing” or “lyophilization” refers to the freeze-drying of a substance by freezing it and then reducing the surrounding pressure (e.g., below 15 Pa, such as below 10 Pa, below 5 Pa, or 1 Pa or less) to allow the frozen medium in the substance to sublimate directly from the solid phase to the gas phase. Thus, the terms “lyophilizing” and “freeze-drying” are used herein interchangeably.
The term “recombinant” in the context of the present disclosure means “made through genetic engineering”. In one embodiment, a “recombinant object” in the context of the present disclosure is not occurring naturally.
The term “naturally occurring” as used herein refers to the fact that an object can be found in nature. For example, a peptide or nucleic acid that is present in an organism (including viruses) and can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally occurring. The term “found in nature” means “present in nature” and includes known objects as well as objects that have not yet been discovered and/or isolated from nature, but that may be discovered and/or isolated in the future from a natural source.
According to the present disclosure, the term “peptide” comprises oligo- and polypeptides and refers to substances which comprise about two or more, about 3 or more, about 4 or more, about 6 or more, about 8 or more, about 10 or more, about 13 or more, about 16 or more, about 20 or more, and up to about 50, about 100 or about 150, consecutive amino acids linked to one another via peptide bonds. The term “protein” refers to large peptides, in particular peptides having at least about 151 amino acids, but the terms “peptide” and “protein” are used herein usually as synonyms.
A “therapeutic protein” has a positive or advantageous effect on a condition or disease state of a subject when provided to the subject in a therapeutically effective amount. In one embodiment, a therapeutic protein has curative or palliative properties and may be administered to ameliorate, relieve, alleviate, reverse, delay onset of or lessen the severity of one or more symptoms of a disease or disorder. A therapeutic protein may have prophylactic properties and may be used to delay the onset of a disease or to lessen the severity of such disease or pathological condition. The term “therapeutic protein” includes entire proteins or peptides and can also refer to therapeutically active fragments thereof. It can also include therapeutically active variants of a protein. Examples of therapeutically active proteins include, but are not limited to, antigens for vaccination and immunostimulants such as cytokines.
The term “portion” refers to a fraction. With respect to a particular structure such as an amino acid sequence or protein the term “portion” thereof may designate a continuous or a discontinuous fraction of said structure.
The terms “part” and “fragment” are used interchangeably herein and refer to a continuous element. For example, a part of a structure such as an amino acid sequence or protein refers to a continuous element of said structure. When used in context of a composition, the term “part” means a portion of the composition. For example, a part of a composition may any portion from 0.1% to 99.9% (such as 0.1%, 0.5%, 1%, 5%, 10%, 50%, 90%, or 99%) of said composition.
“Fragment”, with reference to an amino acid sequence (peptide or protein), relates to a part of an amino acid sequence, i.e. a sequence which represents the amino acid sequence shortened at the N-terminus and/or C-terminus. A fragment shortened at the C-terminus (N-terminal fragment) is obtainable, e.g., by translation of a truncated open reading frame that lacks the 3′-end of the open reading frame. A fragment shortened at the N-terminus (C-terminal fragment) is obtainable, e.g., by translation of a truncated open reading frame that lacks the 5′-end of the open reading frame, as long as the truncated open reading frame comprises a start codon that serves to initiate translation. A fragment of an amino acid sequence comprises, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90% of the amino acid residues from an amino acid sequence. A fragment of an amino acid sequence preferably comprises at least 6, in particular at least 8, at least 12, at least 15, at least 20, at least 30, at least 50, or at least 100 consecutive amino acids from an amino acid sequence.
According to the present disclosure, a part or fragment of a peptide or protein preferably has at least one functional property of the peptide or protein from which it has been derived. Such functional properties comprise a pharmacological activity, the interaction with other peptides or proteins, an enzymatic activity, the interaction with antibodies, and the selective binding of nucleic acids. E.g., a pharmacological active fragment of a peptide or protein has at least one of the pharmacological activities of the peptide or protein from which the fragment has been derived. A part or fragment of a peptide or protein preferably comprises a sequence of at least 6, in particular at least 8, at least 10, at least 12, at least 15, at least 20, at least 30 or at least 50, consecutive amino acids of the peptide or protein. A part or fragment of a peptide or protein preferably comprises a sequence of up to 8, in particular up to 10, up to 12, up to 15, up to 20, up to 30 or up to 55, consecutive amino acids of the peptide or protein.
By “variant” herein is meant an amino acid sequence that differs from a parent amino acid sequence by virtue of at least one amino acid modification. The parent amino acid sequence may be a naturally occurring or wild type (WT) amino acid sequence, or may be a modified version of a wild type amino acid sequence. Preferably, the variant amino acid sequence has at least one amino acid modification compared to the parent amino acid sequence, e.g., from 1 to about 20 amino acid modifications, and preferably from 1 to about 10 or from 1 to about 5 amino acid modifications compared to the parent.
By “wild type” or “WT” or “native” herein is meant an amino acid sequence that is found in nature, including allelic variations. A wild type amino acid sequence, peptide or protein has an amino acid sequence that has not been intentionally modified.
Preferably the degree of similarity, preferably identity between a given amino acid sequence and an amino acid sequence which is a variant of said given amino acid sequence will be at least about 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. The degree of similarity or identity is given preferably for an amino acid region which is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% of the entire length of the reference amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, the degree of similarity or identity is given preferably for at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 amino acids, in some embodiments continuous amino acids. In some embodiments, the degree of similarity or identity is given for the entire length of the reference amino acid sequence. The alignment for determining sequence similarity, preferably sequence identity can be done with art known tools, preferably using the best sequence alignment, for example, using Align, using standard settings, preferably EMBOSS::needle, Matrix: Blosum62, Gap Open 10.0, Gap Extend 0.5.
“Sequence similarity” indicates the percentage of amino acids that either are identical or that represent conservative amino acid substitutions. “Sequence identity” between two amino acid sequences indicates the percentage of amino acids that are identical between the sequences. “Sequence identity” between two nucleic acid sequences indicates the percentage of nucleotides that are identical between the sequences.
The terms “% identical” and “% identity” or similar terms are intended to refer, in particular, to the percentage of nucleotides or amino acids which are identical in an optimal alignment between the sequences to be compared. Said percentage is purely statistical, and the differences between the two sequences may be but are not necessarily randomly distributed over the entire length of the sequences to be compared. Comparisons of two sequences are usually carried out by comparing the sequences, after optimal alignment, with respect to a segment or “window of comparison”, in order to identify local regions of corresponding sequences. The optimal alignment for a comparison may be carried out manually or with the aid of the local homology algorithm by Smith and Waterman, 1981, Ads App. Math. 2, 482, with the aid of the local homology algorithm by Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, with the aid of the similarity search algorithm by Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 88, 2444, or with the aid of computer programs using said algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.). In some embodiments, percent identity of two sequences is determined using the BLASTN or BLASTP algorithm, as available on the United States National Center for Biotechnology Information (NCBI) website (e.g., at blast.ncbi.nlm.nih.gov/Blast.cgi). In some embodiments, the algorithm parameters used for BLASTN algorithm on the NCBI website include: (i) Expect Threshold set to 10; (ii) Word Size set to 28; (iii) Max matches in a query range set to 0; (iv) Match/Mismatch Scores set to 1, -2; (v) Gap Costs set to Linear; and (vi) the filter for low complexity regions being used. In some embodiments, the algorithm parameters used for BLASTP algorithm on the NCBI website include: (i) Expect Threshold set to 10; (ii) Word Size set to 3; (iii) Max matches in a query range set to 0; (iv) Matrix set to BLOSUM62; (v) Gap Costs set to Existence: 11 Extension: 1; and (vi) conditional compositional score matrix adjustment.
Percentage identity is obtained by determining the number of identical positions at which the sequences to be compared correspond, dividing this number by the number of positions compared (e.g., the number of positions in the reference sequence) and multiplying this result by 100.
In some embodiments, the degree of similarity or identity is given for a region which is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% of the entire length of the reference sequence. For example, if the reference amino acid sequence consists of 200 amino acid residues, the degree of identity is given for at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 amino acid residues, in some embodiments continuous amino acid residues. In some embodiments, the degree of similarity or identity is given for the entire length of the reference sequence.
Homologous amino acid sequences exhibit according to the present disclosure at least 40%, in particular at least 50%, at least 60%, at least 70%, at least 80%, at least 90% and preferably at least 95%, at least 98 or at least 99% identity of the amino acid residues.
The amino acid sequence variants described herein may readily be prepared by the skilled person, for example, by recombinant DNA manipulation. The manipulation of DNA sequences for preparing peptides or proteins having substitutions, additions, insertions or deletions, is described in detail in Sambrook et al. (1989), for example. Furthermore, the peptides and amino acid variants described herein may be readily prepared with the aid of known peptide synthesis techniques such as, for example, by solid phase synthesis and similar methods.
In one embodiment, a fragment or variant of an amino acid sequence (peptide or protein) is preferably a “functional fragment” or “functional variant”. The term “functional fragment” or “functional variant” of an amino acid sequence relates to any fragment or variant exhibiting one or more functional properties identical or similar to those of the amino acid sequence from which it is derived, i.e., it is functionally equivalent. With respect to antigens or antigenic sequences, one particular function is one or more immunogenic activities displayed by the amino acid sequence from which the fragment or variant is derived. The term “functional fragment” or “functional variant”, as used herein, in particular refers to a variant molecule or sequence that comprises an amino acid sequence that is altered by one or more amino acids compared to the amino acid sequence of the parent molecule or sequence and that is still capable of fulfilling one or more of the functions of the parent molecule or sequence, e.g., inducing an immune response. In one embodiment, the modifications in the amino acid sequence of the parent molecule or sequence do not significantly affect or alter the characteristics of the molecule or sequence. In different embodiments, the function of the functional fragment or functional variant may be reduced but still significantly present, e.g., immunogenicity of the functional variant may be at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the parent molecule or sequence. However, in other embodiments, immunogenicity of the functional fragment or functional variant may be enhanced compared to the parent molecule or sequence.
An amino acid sequence (peptide, protein or polypeptide) “derived from” a designated amino acid sequence (peptide, protein or polypeptide) refers to the origin of the first amino acid sequence. Preferably, the amino acid sequence which is derived from a particular amino acid sequence has an amino acid sequence that is identical, essentially identical or homologous to that particular sequence or a fragment thereof. Amino acid sequences derived from a particular amino acid sequence may be variants of that particular sequence or a fragment thereof. For example, it will be understood by one of ordinary skill in the art that the antigens suitable for use herein may be altered such that they vary in sequence from the naturally occurring or native sequences from which they were derived, while retaining the desirable activity of the native sequences.
“Isolated” means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not “isolated”, but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated”. An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell. In a preferred embodiment, the binding agent used in the present disclosure is in substantially purified form.
The term “genetic modification” or simply “modification” includes the transfection of cells with nucleic acid. The term “transfection” relates to the introduction of nucleic acids, in particular RNA, into a cell. For purposes of the present disclosure, the term “transfection” also includes the introduction of a nucleic acid into a cell or the uptake of a nucleic acid by such cell, wherein the cell may be present in a subject, e.g., a patient. Thus, according to the present disclosure, a cell for transfection of a nucleic acid described herein can be present in vitro or in vivo, e.g. the cell can form part of an organ, a tissue and/or an organism of a patient. According to the present disclosure, transfection can be transient or stable. For some applications of transfection, it is sufficient if the transfected genetic material is only transiently expressed. RNA can be transfected into cells to transiently express its coded protein. Since the nucleic acid introduced in the transfection process is usually not integrated into the nuclear genome, the foreign nucleic acid will be diluted through mitosis or degraded. Cells allowing episomal amplification of nucleic acids greatly reduce the rate of dilution. If it is desired that the transfected nucleic acid actually remains in the genome of the cell and its daughter cells, a stable transfection must occur. Such stable transfection can be achieved by using virus-based systems or transposon-based systems for transfection. Generally, nucleic acid encoding antigen is transiently transfected into cells. RNA can be transfected into cells to transiently express its coded protein.
According to the present disclosure, an analog of a peptide or protein is a modified form of said peptide or protein from which it has been derived and has at least one functional property of said peptide or protein. E.g., a pharmacological active analog of a peptide or protein has at least one of the pharmacological activities of the peptide or protein from which the analog has been derived. Such modifications include any chemical modification and comprise single or multiple substitutions, deletions and/or additions of any molecules associated with the protein or peptide, such as carbohydrates, lipids and/or proteins or peptides. In one embodiment, “analogs” of proteins or peptides include those modified forms resulting from glycosylation, acetylation, phosphorylation, amidation, palmitoylation, myristoylation, isoprenylation, lipidation, alkylation, derivatization, introduction of protective/blocking groups, proteolytic cleavage or binding to an antibody or to another cellular ligand. The term “analog” also extends to all functional chemical equivalents of said proteins and peptides.
“Activation” or “stimulation”, as used herein, refers to the state of an immune effector cell such as T cell that has been sufficiently stimulated to induce detectable cellular proliferation. Activation can also be associated with initiation of signaling pathways, induced cytokine production, and detectable effector functions. The term “activated immune effector cells” refers to, among other things, immune effector cells that are undergoing cell division.
The term “priming” refers to a process wherein an immune effector cell such as a T cell has its first contact with its specific antigen and causes differentiation into effector cells such as effector T cells.
The term “clonal expansion” or “expansion” refers to a process wherein a specific entity is multiplied. In the context of the present disclosure, the term is preferably used in the context of an immunological response in which immune effector cells are stimulated by an antigen, proliferate, and the specific immune effector cell recognizing said antigen is amplified. Preferably, clonal expansion leads to differentiation of the immune effector cells.
An “antigen” according to the present disclosure covers any substance that will elicit an immune response and/or any substance against which an immune response or an immune mechanism such as a cellular response is directed. This also includes situations wherein the antigen is processed into antigen peptides and an immune response or an immune mechanism is directed against one or more antigen peptides, in particular if presented in the context of MHC molecules. In particular, an “antigen” relates to any substance, preferably a peptide or protein, that reacts specifically with antibodies or T-lymphocytes (T-cells). According to the present disclosure, the term “antigen” comprises any molecule which comprises at least one epitope, such as a T cell epitope. Preferably, an antigen in the context of the present disclosure is a molecule which, optionally after processing, induces an immune reaction, which is preferably specific for the antigen (including cells expressing the antigen). In one embodiment, an antigen is a disease-associated antigen, such as a tumor antigen, a viral antigen, or a bacterial antigen, or an epitope derived from such antigen.
The term “epitope” refers to an antigenic determinant in a molecule such as an antigen, i.e., to a part in or fragment of the molecule that is recognized by the immune system, for example, that is recognized by antibodies T cells or B cells, in particular when presented in the context of MHC molecules. In one embodiment, “epitope” means a protein determinant capable of specific binding to an antibody. Epitopes usually consist of surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents. The epitope may comprise amino acid residues directly involved in the binding and other amino acid residues, which are not directly involved in the binding, such as amino acid residues which are effectively blocked or covered by the specifically antigen-binding peptide (in other words, the amino acid residue is within the footprint of the specifically antigen-binding peptide).
An epitope of a protein preferably comprises a continuous or discontinuous portion of said protein and is preferably between about 5 and about 100, preferably between about 5 and about 50, more preferably between about 8 and about 0, most preferably between about 10 and about 25 amino acids in length, for example, the epitope may be preferably 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. It is particularly preferred that the epitope in the context of the present disclosure is a T cell epitope.
The term “optional” or “optionally” as used herein means that the subsequently described event, circumstance or condition may or may not occur, and that the description includes instances where said event, circumstance, or condition occurs and instances in which it does not occur.
As used herein, the terms “linked”, “fused”, or “fusion” are used interchangeably. These terms refer to the joining together of two or more elements or components or domains.
The term “disease” (also referred to as “disorder” herein) refers to an abnormal condition that affects the body of an individual. A disease is often construed as a medical condition associated with specific symptoms and signs. A disease may be caused by factors originally from an external source, such as infectious disease, or it may be caused by internal dysfunctions, such as autoimmune diseases. In humans, “disease” is often used more broadly to refer to any condition that causes pain, dysfunction, distress, social problems, or death to the individual afflicted, or similar problems for those in contact with the individual. In this broader sense, it sometimes includes injuries, disabilities, disorders, syndromes, infections, isolated symptoms, deviant behaviors, and atypical variations of structure and function, while in other contexts and for other purposes these may be considered distinguishable categories. Diseases usually affect individuals not only physically, but also emotionally, as contracting and living with many diseases can alter one's perspective on life, and one's personality.
The term “therapeutic treatment” relates to any treatment which improves the health status and/or prolongs (increases) the lifespan of an individual. Said treatment may eliminate the disease in an individual, arrest or slow the development of a disease in an individual, inhibit or slow the development of a disease in an individual, decrease the frequency or severity of symptoms in an individual, and/or decrease the recurrence in an individual who currently has or who previously has had a disease.
The terms “prophylactic treatment” or “preventive treatment” relate to any treatment that is intended to prevent a disease from occurring in an individual. The terms “prophylactic treatment” or “preventive treatment” are used herein interchangeably. Similarly, the term “method for preventing” in the context of progression of a disease, such as progression of a tumor or cancer, relates to any method that is intended to prevent the disease from progressing in an individual.
The terms “individual” and “subject” are used herein interchangeably. They refer to a human or another mammal (e.g., mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate), or any other non-mammal-animal, including birds (chicken), fish or any other animal species that can be afflicted with or is susceptible to a disease or disorder (e.g., cancer). Unless otherwise stated, the terms “individual” and “subject” do not denote a particular age, and thus encompass adults, elderlies, children, and newborns. In embodiments of the present disclosure, the “individual” or “subject” is a “patient”.
The term “patient” means an individual or subject for treatment, in particular a diseased individual or subject.
In a first aspect, the present disclosure provides a binding agent for use in a method for reducing or preventing progression of a tumor or treating cancer in a subject, said method comprising administering to said subject the binding agent prior to, simultaneously with, or after administration of an antibody binding to Programmed Death-1 (PD-1), or an antigen-binding fragment thereof, wherein the binding agent comprises a first binding region binding to CD137 and a second binding region binding to PD-L1,
In one embodiment, CD137 is human CD137, in particular human CD137 comprising the sequence set forth in SEQ ID NO: 38. In one embodiment, PD-L1 is human PD-L1, in particular human PD-L1 comprising the sequence set forth in SEQ ID NO: 40. In one embodiment, CD137 is human CD137 and PD-L1 is human PD-L1. In one embodiment, CD137 is human CD137 comprising the sequence set forth in SEQ ID NO: 38, and PD-L1 is human PD-L1 comprising the sequence set forth in SEQ ID NO: 40.
In one embodiment of the binding agent according to the first aspect, the first binding region binding to human CD137 comprises a heavy chain variable region (VH) comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to SEQ ID NO: 1 or 9 and a light chain variable region (VL) region and comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to SEQ ID NO: 5 or 10.
In further embodiment of the binding agent according to the first aspect, the second binding region binding to human PD-L1 comprises a heavy chain variable region (VH) comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 25 100% sequence identity to SEQ ID NO: 11 and a light chain variable region (VL) region comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to SEQ ID NO: 15.
In one embodiment of the binding agent according to the first aspect,
In one embodiment of the binding agent according to the first aspect, the first binding region binding to human CD137 comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 1 or 9 and a light chain variable region (VL) region comprising the amino acid sequence set forth in SEQ ID NO: 5 or 10.
In a further embodiment of the binding agent according to the first aspect, the second binding region binding to human PD-L1 comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 11 and a light chain variable region (VL) region comprising the amino acid sequence set forth in SEQ ID NO: 15.
In one embodiment of the binding agent according to the first aspect,
In one embodiment of the binding agent according to the first aspect,
The binding agent may in particular be an antibody, such as a multispecific antibody, e.g., a bispecific antibody. Also, the binding agent may be in the format of a full-length antibody or an antibody fragment.
It is further preferred that the binding agent is a human antibody or a humanized antibody.
Each variable region may comprise three complementarity determining regions (CDR1, CDR2, and CDR3) and four framework regions (FR1, FR2, FR3, and FR4).
The complementarity determining regions (CDRs) and the framework regions (FRs) may be arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
In one embodiment of the first aspect, the binding agent comprises
In one embodiment of the first aspect, the binding agent comprises
In one embodiment of the first aspect, the binding agent is an antibody comprising a first binding arm and a second binding arm, wherein the first binding arm comprises
In one embodiment of the first aspect, the binding agent comprises i) a first heavy chain and light chain comprising said antigen-binding region capable of binding to CD137, the first heavy chain comprising a first heavy chain constant region and the first light chain comprising a first light chain constant region; and ii) a second heavy chain and light chain comprising said antigen-binding region capable of binding PD-L1, the second heavy chain comprising a second heavy chain constant region and the second light chain comprising a second light chain constant region.
Each of the first and second heavy chain constant regions (CH) may comprise one or more of a constant heavy chain 1 (CH1) region, a hinge region, a constant heavy chain 2 (CH2) region and a constant heavy chain 3 (CH3) region, preferably at least a hinge region, a CH2 region and a CH3 region.
Each of the first and second heavy chain constant regions (CHs) may comprise a CH3 region, wherein the two CH3 regions comprise asymmetrical mutations. Asymmetrical mutations mean that the sequences of said first and second CH3 regions contain amino acid substitutions at non-identical positions. For example, one of said first and second CH3 regions contains a mutation at the position corresponding to position 405 in a human IgG1 heavy chain according to EU numbering, and the other of said first and second CH3 regions contains a mutation at the position corresponding to position 409 in a human IgG1 heavy chain according to EU numbering.
In said first heavy chain constant region (CH) at least one of the amino acids in a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in a human IgG1 heavy chain according to EU numbering may have been substituted, and in said second heavy chain constant region (CH) at least one of the amino acids in a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in a human IgG1 heavy chain according to EU numbering may have been substituted. In particular embodiments, the first and the second heavy chains are not substituted in the same positions (i.e., the first and the second heavy chains contain asymmetrical mutations).
In one embodiment of the binding agent according to the first aspect, (i) the amino acid in the position corresponding to F405 in a human IgG1 heavy chain according to EU numbering is L in said first heavy chain constant region (CH), and the amino acid in the position corresponding to K409 in a human IgG1 heavy chain according to EU numbering is R in said second heavy chain constant region (CH), or (ii) the amino acid in the position corresponding to K409 in a human IgG1 heavy chain according to EU numbering is R in said first heavy chain, and the amino acid in the position corresponding to F405 in a human IgG1 heavy chain according to EU numbering is L in said second heavy chain.
In one embodiment of the first aspect, the binding agent induces Fc-mediated effector function to a lesser extent compared to another antibody comprising the same first and second antigen binding regions and two heavy chain constant regions (CHs) comprising human IgG1 hinge, CH2 and CH3 regions.
In one particular embodiment of the binding agent according to the first aspect, said first and second heavy chain constant regions (CHs) are modified so that the antibody induces Fc-mediated effector function to a lesser extent compared to an antibody which is identical except for comprising non-modified first and second heavy chain constant regions (CHs). In particular, each or both of said non-modified first and second heavy chain constant regions (CHs) may comprise, consists of or consist essentially of the amino acid sequence set forth in SEQ ID NO: 19 or 25.
The Fc-mediated effector function may be determined by measuring binding of the binding agent to Fcγ receptors, binding to C1q, or induction of Fc-mediated cross-linking of Fcγ receptors. In particular, the Fc-mediated effector function may be determined by measuring binding of the binding agent to C1q.
The first and second heavy chain constant regions of the binding agent may have been modified so that binding of C1q to said antibody is reduced compared to a wild-type antibody, preferably reduced by at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100%, wherein C1q binding is preferably determined by ELISA.
In one embodiment of the binding agent according to the first aspect, in at least one of said first and second heavy chain constant regions (CH), one or more amino acids in the positions corresponding to positions L234, L235, D265, N297, and P331 in a human IgG1 heavy chain according to EU numbering, are not L, L, D, N, and P, respectively.
In one embodiment of the binding agent according to the first aspect, the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain according to EU numbering may be F and E, respectively, in said first and second heavy chains.
In particular, the positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain according to EU numbering may be F, E, and A, respectively, in said first and second heavy chain constant regions (HCs).
In one embodiment of the binding agent according to the first aspect, the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain according to EU numbering of both the first and second heavy chain constant regions are F and E, respectively, wherein (i) the position corresponding to F405 in a human IgG1 heavy chain according to EU numbering of the first heavy chain constant region is L, and the position corresponding to K409 in a human IgG1 heavy chain according to EU numbering of the second heavy chain is R, or (ii) the position corresponding to K409 in a human IgG1 heavy chain according to EU numbering of the first heavy chain constant region is R, and the position corresponding to F405 in a human IgG1 heavy chain according to EU numbering of the second heavy chain is L.
In one embodiment of the binding agent according to the first aspect, the positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain according to EU numbering of both the first and second heavy chain constant regions are F, E, and A, respectively, wherein (i) the position corresponding to F405 in a human IgG1 heavy chain according to EU numbering of the first heavy chain constant region is L, and the position corresponding to K409 in a human IgG1 heavy chain according to EU numbering of the second heavy chain constant region is R, or (ii) the position corresponding to K409 in a human IgG1 heavy chain according to EU numbering of the first heavy chain is R, and the position corresponding to F405 in a human IgG1 heavy chain according to EU numbering of the second heavy chain is L.
In one embodiment of the binding agent according to the first aspect, the constant region of said first and/or second heavy chain comprises an amino acid sequence selected from the group consisting of
In one embodiment of the binding agent according to the first aspect, the constant region of said first or second heavy chain, such as the second heavy chain, comprises or consists essentially of or consists of an amino acid sequence selected from the group consisting of
In one embodiment of the binding agent according to the first aspect, the constant region of said first or second heavy chain, such as the first heavy chain comprises or consists essentially of or consists of an amino acid sequence selected from the group consisting of
In one embodiment of the binding agent according to the first aspect, the constant region of said first and/or second heavy chain comprises or consists essentially of or consists of an amino acid sequence selected from the group consisting of
In one embodiment of the binding agent according to the first aspect, the constant region of said first and/or second heavy chain, such as the second heavy chain, comprises or consists essentially of or consists of an amino acid sequence selected from the group consisting of
In one embodiment of the binding agent according to the first aspect, the constant region of said first and/or second heavy chain, such as the first heavy chain, comprises or consists essentially of or consists of an amino acid sequence selected from the group consisting of
In one embodiment of the first aspect, the binding agent comprises a kappa (κ) light chain constant region.
In one embodiment of the first aspect, the binding agent comprises a lambda (λ) light chain constant region.
In one embodiment of the binding agent according to the first aspect, the first light chain constant region is a kappa (κ) light chain constant region or a lambda (λ) light chain constant region.
In one embodiment of the binding agent according to the first aspect, the second light chain constant region is a lambda (λ) light chain constant region or a kappa (κ) light chain constant region.
In one embodiment of the binding agent according to the first aspect, the first light chain constant region is a kappa (κ) light chain constant region and the second light chain constant region is a lambda (λ) light chain constant region or the first light chain constant region is a lambda (λ) light chain constant region and the second light chain constant region is a kappa (κ) light chain constant region.
In one embodiment of the binding agent according to the first aspect, the kappa (κ) light chain comprises an amino acid sequence selected from the group consisting of
In one embodiment of the binding agent according to the first aspect, the lambda (λ) light chain comprises an amino acid sequence selected from the group consisting of
The binding agent (in particular, antibody) according to the first aspect is of an isotype selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. In particular, the binding agent may be a full-length IgG1 antibody. In preferred embodiments of the first aspect, the binding agent (in particular, antibody) is of the IgG1m(f) allotype.
In a preferred embodiment of the binding agent according to the first aspect, the binding agent comprises
The binding agent for use according to the first aspect may in particular be acasunlimab or a biosimilar thereof.
In currently preferred embodiments, the amount of binding agent administered in each dose and/or in each treatment cycle is
According to these embodiments, the dose defined in mg/kg may be converted to flat dose, and vice versa, based on the median body weight of the subjects to whom the binding agent is administered being 80 kg
The amount of binding agent administered in each dose and/or in each treatment cycle may in particular be about 0.3-4.0 mg/kg body weight or about 25-320 mg in total; and/or about 2.1×10−9-2.7×10−8 mol/kg body weight or about 1.7×10−7-2.2×10−6 mol in total.
The amount of binding agent administered in each dose and/or in each treatment cycle may in particular be about 0.38-4.0 mg/kg body weight or about 30-320 mg in total; and/or about 2.6×10−9-2.7×10−8 mol/kg body weight or about 2.4×10−7-2.2×10−6 mol in total.
The amount of binding agent administered in each dose and/or in each treatment cycle may in particular be about 0.5-3.3 mg/kg body weight or about 40-260 mg in total; and/or about 3.4×10−9-2.2×10−8 mol/kg body weight or about 2.7×10−7-1.8×10−6 mol in total.
The amount of binding agent administered in each dose and/or in each treatment cycle may in particular be about 0.6-2.5 mg/kg body weight or about 50-200 mg in total; and/or about 4.3×10−9-1.7×10−8 mol/kg body weight or about 3.4×10−7-1.4×10−6 mol in total.
The amount of binding agent administered in each dose and/or in each treatment cycle may in particular be about 0.8-1.8 mg/kg body weight or about 60-140 mg in total; and/or about 5.1×10−9-1.2×10−8 mol/kg body weight or about 4.1×10−7-9.5×10−7 mol in total.
The amount of binding agent administered in each dose and/or in each treatment cycle may in particular be about 0.9-1.8 mg/kg body weight or about 70-140 mg in total; and/or about 6.0×10−9-1.2×10−8 mol/kg body weight or about 4.8×10−7-9.5×10−7 mol in total.
The amount of binding agent administered in each dose and/or in each treatment cycle may in particular be about 1-1.5 mg/kg body weight or about 80-120 mg in total; and/or about 6.8×10−9-1.0×10−8 mol/kg body weight or about 5.5×10−7-8.2×10−7 mol in total.
The amount of binding agent administered in each dose and/or in each treatment cycle may in particular be about 1.1-1.4 mg/kg body weight or about 90-110 mg in total; and/or about 7.7×10−9-9.4×10−9 mol/kg body weight or about 6.1×10−7-7.5×10−7 mol in total.
The amount of binding agent administered in each dose and/or in each treatment cycle may in particular be about 1.2-1.3 mg/kg body weight or about 95-105 mg in total; and/or about 6.8×10−9-8.9×10−9 mol/kg body weight or about 6.5×10−7-7.2×10−7 mol in total.
The amount of binding agent administered in each dose and/or in each treatment cycle may in particular be about 0.8-1.5 mg/kg body weight or about 65-120 mg in total; and/or about 5.5×10−9-1.0×10−8 mol/kg body weight or about 4.4×10−7-8.2×10−7 mol in total.
The amount of binding agent administered in each dose and/or in each treatment cycle may in particular be about 0.9-1.3 mg/kg body weight or about 70-100 mg in total; and/or about 6.0×10−9-8.5×10−9 mol/kg body weight or about 4.8×10−7-6.8×10−7 mol in total. about 0.9-1.1 mg/kg body weight or about 75-90 mg in total; and/or about 6.4×10−9-7.7×10−9 mol/kg body weight or about 5.1×10−7-6.1×10−7 mol in total.
Further, the amount of binding agent administered in each dose and/or in each treatment cycle may in particular be 0.3-4.0 mg/kg body weight or 25-320 mg in total; and/or 2.1×10−9-2.7×10−8 mol/kg body weight or 1.7×10−7-2.2×10−7 mol in total.
The amount of binding agent administered in each dose and/or in each treatment cycle may in particular be 0.38-4.0 mg/kg body weight or 30-320 mg in total; and/or 2.6×10−9-2.7×10−8 mol/kg body weight or 2.4×10−7-2.2×10−7 mol in total.
The amount of binding agent administered in each dose and/or in each treatment cycle may in particular be 0.5-3.3 mg/kg body weight or 40-260 mg in total; and/or 3.4×10−9-2.2×10−8 mol/kg body weight or 2.7×10−7-1.8×10−7 mol in total.
The amount of binding agent administered in each dose and/or in each treatment cycle may in particular be 0.6-2.5 mg/kg body weight or 50-200 mg in total; and/or 4.3×10−9-1.7×10−8 mol/kg body weight or 3.4×10−7-1.4×10−6 mol in total.
The amount of binding agent administered in each dose and/or in each treatment cycle may in particular be 0.8-1.8 mg/kg body weight or 60-140 mg in total; and/or 5.1×10−9-1.2×10−8 mol/kg body weight or 4.1×10−7-9.5×10−7 mol in total.
The amount of binding agent administered in each dose and/or in each treatment cycle may in particular be 0.9-1.8 mg/kg body weight or 70-140 mg in total; and/or 6.0×10−9-1.2×10−8 mol/kg body weight or 4.8×10−7-9.5×10−7 mol in total.
The amount of binding agent administered in each dose and/or in each treatment cycle may in particular be 1-1.5 mg/kg body weight or 80-120 mg in total; and/or 6.8×10−9-1.0×10−8 mol/kg body weight or 5.5×10−7-8.2×10−7 mol in total.
The amount of binding agent administered in each dose and/or in each treatment cycle may in particular be 1.1-1.4 mg/kg body weight or 90-110 mg in total; and/or 7.7×10−9-9.4×10−9 mol/kg body weight or 6.1×10−7-7.5×10−7 mol in total.
The amount of binding agent administered in each dose and/or in each treatment cycle may in particular be 1.2-1.3 mg/kg body weight or 95-105 mg in total; and/or 6.8×10−9-8.9×10−9 mol/kg body weight or 6.5×10−7-7.2×10−7 mol in total.
The amount of binding agent administered in each dose and/or in each treatment cycle may in particular be 0.8-1.5 mg/kg body weight or 65-120 mg in total; and/or 5.5×10−9-1.0×10−8 mol/kg body weight or 4.4×10−7-8.2×10−7 mol in total.
The amount of binding agent administered in each dose and/or in each treatment cycle may in particular be 0.9-1.3 mg/kg body weight or 70-100 mg in total; and/or 6.0×10−9-8.5×10−9 mol/kg body weight or 4.8×10−7-6.8×10−7 mol in total.
The amount of binding agent administered in each dose and/or in each treatment cycle may in particular be 0.9-1.1 mg/kg body weight or 75-90 mg in total; and/or 6.4×10−9-7.7×10−9 mol/kg body weight or 5.1×10−7-6.1×10−7 mol in total.
The amount of binding agent administered in each dose and/or in each treatment cycle may be
The amount of binding agent administered in each dose and/or in each treatment cycle may be
It is currently preferred that the amount of binding agent administered in each dose and/or in each treatment cycle is
It is equally preferred that the amount of binding agent administered in each dose and/or in each treatment cycle is
The binding agent may be administered in any manner and by any route known in the art. In a preferred embodiment, the binding agent is administered systemically, such as parenterally, in particular intravenously.
The binding agent may be administered in the form of any suitable pharmaceutical composition as described herein. In a preferred embodiment, the binding agent is administered in the form of an infusion.
The binding agent for use according to the invention may be administered by using intravenous (IV) infusion, such as by intravenous infusion over a minimum of 30 minutes, such as over a minimum of 60 minutes e.g., by using intravenous infusion over 30 to 120 minutes. Preferably, the binding agent for use according to the invention is administered by using intravenous (IV) infusion over 30 minutes.
The binding agent can be administered prior to, simultaneously with, or after administration of the PD-1 inhibitor.
In one embodiment, the binding agent is administered prior to the administration of the PD-1 inhibitor. For example, the gap between the end of the administration of the binding agent and the beginning of the administration of the PD-1 inhibitor can be at least about 10 min, such as at least about 15 min, at least about 20 min, at least about 25 min, at least about 30 min, at least about 35 min, at least about 40 min, at least about 45 min, at least about 50 min, at least about 55 min, at least about 60 min, at least about 90 min, or at least about 120 min, and up to about 14 days (up to about 2 weeks), such as up to about 13 days, up to about 12 days, up to about 11 days, up to about 10 days, up to about 9 days, up to about 8 days, up to about 7 days (up to about 1 week), up to about 6 days, up to about 5 days, up to about 4 days, up to about 3 days, up to about 2 days, up to about 1 day (up to about 24 h), up to about 18 h, up to about 12 h, up to about 6 h, up to about 5 h, up to about 4 h, up to about 3 h, up to about 2.5 h, or up to about 2 h.
In one embodiment, the binding agent is administered after the administration of the PD-1 inhibitor. For example, the gap between the end of the administration of the PD-1 inhibitor and the beginning of the administration of the binding agent can be at least about 10 min, such as at least about 15 min, at least about 20 min, at least about 25 min, at least about 30 min, at least about 35 min, at least about 40 min, at least about 45 min, at least about 50 min, at least about 55 min, at least about 60 min, at least about 90 min, or at least about 120 min, and up to about 14 days (up to about 2 weeks), such as up to about 13 days, up to about 12 days, up to about 11 days, up to about 10 days, up to about 9 days, up to about 8 days, up to about 7 days (up to about 1 week), up to about 6 days, up to about 5 days, up to about 4 days, up to about 3 days, up to about 2 days, up to about 1 day (up to about 24 h), up to about 18 h, up to about 12 h, up to about 6 h, up to about 5 h, up to about 4 h, up to about 3 h, up to about 2.5 h, or up to about 2 h.
In one embodiment, the binding agent is administered simultaneously with the PD-1 inhibitor. For example, the binding agent and the PD-1 inhibitor may be administered using a composition comprising both drugs. Alternatively, the binding agent may be administered into one extremity of the subject, and the PD-1 inhibitor may be administered into another extremity of the subject.
The antibody binding to PD-1 or the antigen-binding fragment thereof preferably comprises a heavy chain variable region comprising an amino acid sequence having at least 85% sequence identity, such as at least 90% sequence identity. 95% sequence identity. 98% sequence identity or 99% sequence identity, to the amino acid sequence of SEQ ID NO: 49 and a light chain variable region comprising an amino acid sequence having at least 85% sequence identity, such as at least 90% sequence identity, 95% sequence identity. 98% sequence identity or 99% sequence identity, to the amino acid sequence of SEQ ID NO: 50.
In currently most preferred embodiments, the antibody binding to PD-1 or the antigen-binding fragment thereof comprises a heavy chain variable region comprising, consisting of or consisting essentially of the amino acid sequence of SEQ ID NO: 49 and a light chain variable region comprising, consisting of or consisting essentially of the amino acid sequence of SEQ ID NO: 50.
The antibody binding to PD-1 or the antigen-binding fragment thereof may comprise a heavy chain comprising, consisting of or consisting essentially of the amino acid sequence of SEQ ID NO: 51 and a light chain comprising, consisting of or consisting essentially of the amino acid sequence of SEQ ID NO: 52.
The antibody binding to PD-1 used according to the present invention preferably prevents inhibitory signals associated with PD-1. The antibody binding to PD-1 preferably disrupts or inhibits inhibitory signaling associated with PD-1.
Inhibiting or blocking of PD-1 signaling, as described herein, results in preventing or reversing immune-suppression and establishment or enhancement of T cell immunity against cancer cells. In one embodiment, inhibition of PD-1 signaling, as described herein, reduces or inhibits dysfunction of the immune system. In one embodiment, inhibition of PD-1 signaling, as described herein, renders dysfunctional immune cells less dysfunctional. In one embodiment, inhibition of PD-1 signaling, as described herein, renders a dysfunctional T cell less dysfunctional.
In one embodiment, the PD-1 inhibitor prevents the interaction between PD-1 and PD-L1. In another embodiment, the PD-1 inhibitor prevents the interaction between PD-1 and PD-L2.
In particular, the antibody binding to PD-1 or the antigen-binding fragment thereof is a chimerized, humanized or human antibody.
In a preferred embodiment, the antibody binding to PD-1 is an isolated antibody.
Without being bound by theory the combination of a binding agent comprising a first binding region binding to CD137 and a second binding region binding to PD-L1 as defined above with an antibody binding to PD-1 as defined above is believed to increase the response rate and lead to improved duration of response in subjects receiving the combination therapy because the combination therapy leads to complete blockade of the PD-1 pathway with concurrent conditional activation of 4-1BB. A PD-1 blocking antibody blocks interaction with both PD-L1 and PD-L2. It is further believed that the combination therapy with an antibody binding to PD-1 makes increased amounts of PD-L1 available to be bound by the binding agent.
The PD-1 inhibitor may in particular be pembrolizumab or a biosimilar thereof.
In a further embodiment the PD-1 inhibitor is an antibody comprising a heavy chain variable region (VH) comprising or consisting of or consisting essentially of the sequence set forth in SEQ ID NO: 49, and a light chain variable region (VL) comprising, consisting of or consisting essentially of the sequence set forth in SEQ ID NO: 50. The PD-1 inhibitor may in particular be an antibody comprising a heavy chain comprising, consisting of or consisting essentially of the amino acid sequence set forth in SEQ ID NO: 51, and a light chain comprising, consisting of or consisting essentially of the amino acid sequence set forth in SEQ ID NO: 52.
Anti-PD-1 antibodies of the disclosure are preferably monoclonal, and may be multispecific, human, humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab′) fragments, fragments produced by a Fab expression library, and PD-1 binding fragments of any of the above. In some embodiments, an anti-PD-1 antibody described herein binds specifically to PD-1 (e.g., human PD-1). The immunoglobulin molecules of the disclosure can be of any isotype (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule. antigen-binding fragments (e.g., human antigen-binding fragments) as described herein and include, but are not limited to, Fab, Fab′ and F(ab′)2, Fd, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv) and fragments comprising either a VL or VH domain. Antigen-binding fragments, including single-chain antibodies, may comprise the variable region(s) alone or in combination with the entirety or a portion of the following: hinge region, CH1, CH2, CH3 and CL domains. Also included in the present disclosure are antigen-binding fragments comprising any combination of variable region(s) with a hinge region, CH1, CH2, CH3 and CL domains. In some embodiments, the anti-PD-1 antibodies or antigen-binding fragments thereof are human, murine (e.g., mouse and rat), donkey, sheep, rabbit, goat, guinea pig, camelid, horse, or chicken.
In some embodiments, numbering of amino acid residues in CDR sequences of anti-PD-1 antibodies or antigen-binding fragments thereof provided herein are according to the IMGT numbering scheme as described in Lefranc, M. P. et al., Dev. Comp. Immunol., 2003, 27, 55-77.
The antibody binding to PD-1 or the antigen-binding fragment thereof also include derivatives and constructs that are modified, i.e., by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not prevent the antibody from binding to PD-1. For example, but not by way of limitation, the anti-PD-1 antibody derivatives include antibodies that have been modified, e.g., by glycosylation, acetylation, PEGylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the derivative or construct may contain one or more non-classical amino acids.
Preferably, the antibody binding to PD-1 or the antigen-binding fragment thereof is administered in a suitable amount. The amount of antibody binding to PD-1 or antigen-binding fragment thereof administered in each dose and/or treatment cycle may in particular be in a range, wherein more than 5%, preferably more than 10%, more preferably more than 15%, even more preferably more than 20%, even more preferably more than 25%, even more preferably more than 30%, even more preferably more than 35%, even more preferably more than 40%, even more preferably more than 45%, most preferably more than 50% of said PD-1 inhibitors bind to PD-1.
In certain embodiments, the antibody binding to PD-1 is pembrolizumab or a biosimilar thereof and the amount of PD-1 inhibitor administered, e.g., in each dose and/or in each treatment cycle, is about 10-about 1000 mg in total such as about 100-about 600 mg in total, e.g., about 150-about 600 mg in total, about 150-about 500 mg in total, about 175-about 500 mg in total, about 175-about 450 mg in total, about 200-about 450 mg in total or such as about 200-about 400 mg in total.
In certain embodiments, the antibody binding to PD-1 is pembrolizumab or a biosimilar thereof and the amount of PD-1 inhibitor administered, e.g., in each dose and/or in each treatment cycle, is 10-1000 mg in total such as 100-600 mg in total, e.g., 150-600 mg in total, 150-500 mg in total, 175-500 mg in total, 175-450 mg in total, 200-450 mg in total or such as 200-400 mg in total.
In certain embodiments, the antibody binding to PD-1 is pembrolizumab or a biosimilar thereof and the amount of antibody binding to PD-1 administered, e.g., in each dose and/or in each treatment cycle, is about 100-600 mg in total; and/or about 6.84×10−7-4.11×10−7 mol in total.
In certain embodiments, the antibody binding to PD-1 is pembrolizumab or a biosimilar thereof and the amount of antibody binding to PD-1 administered, e.g., in each dose and/or in each treatment cycle, is about 100-400 mg in total; and/or about 6.84×10−7-2.73×10−6 mol in total, such as 100-400 mg in total; and/or 6.84×10−7-2.73×10−6 mol in total.
In certain embodiments, the antibody binding to PD-1 is pembrolizumab or a biosimilar thereof and the amount of antibody binding to PD-1 administered, e.g., in each dose and/or in each treatment cycle, is about 200-400 mg in total; and/or about 6.84×10−7-2.73×10−6 mol in total, such as 200-400 mg in total; and/or 6.84×10−7-2.73×10−6 mol in total.
In certain embodiments, the amount of antibody binding to PD-1 or antigen-binding fragment thereof administered, e.g., in each dose and/or in each treatment cycle, is about 200 mg or about 1.37×10−6 mol in total, such as 200 mg or 1.37×10−6 mol in total.
In certain embodiments, the antibody binding to PD-1 is pembrolizumab or a biosimilar thereof and the amount of antibody binding to PD-1 or antigen-binding fragment thereof administered, e.g., in each dose and/or in each treatment cycle, is about 200 mg or about 1.37×10−6 mol in total, such as 200 mg or 1.37×10−6 mol in total.
In certain embodiments, the amount of antibody binding to PD-1 or antigen-binding fragment thereof administered, e.g., in each dose and/or in each treatment cycle, is about 400 mg in total or about 2.73×10−6 in total, such as 400 mg in total or 2.73×10−6 in total.
In certain embodiments, the antibody binding to PD-1 is pembrolizumab or a biosimilar thereof and the amount of antibody binding to PD-1 or antigen-binding fragment thereof administered, e.g., in each dose and/or in each treatment cycle, is about 400 mg in total or about 2.73×10−6 in total, such as 400 mg in total or 2.73×10−6 in total.
The antibody binding to PD-1 or antigen-binding fragment thereof may be administered in any manner and by any route known in the art. The mode and route of administration will depend on the type of antibody to be used. In a preferred embodiment, the antibody binding to PD-1 or antigen-binding fragment thereof is administered systemically, such as parenterally, in particular intravenously.
The antibody binding to PD-1 or antigen-binding fragment thereof may be administered in the form of any suitable pharmaceutical composition as described herein. In a preferred embodiment, the antibody binding to PD-1 or antigen-binding fragment thereof is administered in the form of an infusion, such as an intravenous infusion.
The subject to be treated according to the present disclosure is preferably a human subject.
In one preferred embodiment, the tumor or cancer to be treated is a solid tumor or cancer. The tumor or cancer may be a metastatic tumor or cancer.
Preferably, the tumor or cancer may be selected from the group consisting of melanoma, ovarian cancer, lung cancer (e.g., non-small cell lung cancer (NSCLC)), colorectal cancer, head and neck cancer, gastric cancer, breast cancer, renal cancer, urothelial cancer, bladder cancer, esophageal cancer, pancreatic cancer, hepatic cancer, thymoma and thymic carcinoma, brain cancer, glioma, adrenocortical carcinoma, thyroid cancer, other skin cancers, sarcoma, multiple myeloma, leukemia, lymphoma, myelodysplastic syndromes, endometrial cancer, prostate cancer, penile cancer, cervical cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, Merkel cell carcinoma and mesothelioma. More preferably, the tumor or cancer is selected from the group consisting of melanoma, lung cancer, colorectal cancer, pancreatic cancer, and head and neck cancer.
In particular embodiments, the tumor or cancer is selected from the group consisting of lung cancer (e.g. non-small cell lung cancer (NSCLC), urothelial cancer (cancer of the bladder, ureter, urethra, or renal pelvis), endometrial cancer (EC), breast cancer (e.g. triple negative breast cancer (TNBC)), squamous cell carcinoma of the head and neck (SCCHN) (e.g. cancer of the oral cavity, pharynx or larynx) and cervical cancer.
Preferably, the tumor is a PD-L1 positive tumor. In certain embodiments, it is preferred that PD-L1 is expressed in ≥1% of the cancer cells or tumor cells. The expression of PD-L1 may be determined using techniques known to the person skilled in the art and may e.g. be assessed by immunohistochemistry (IHC).
The tumor or cancer may in particular be a lung cancer. The lung cancer may be a non-small cell lung cancer (NSCLC), such as a squamous or a non-squamous NSCLC. Lung cancer is the second most common malignancy with an estimated age-standardized incidence rate of 22.4 per 100,000 and a leading cause of cancer death for both men and women (Kantar, 2021). Worldwide, approximately 2,206,771 new cases of lung cancer and 1,796,144 deaths are estimated in 2020 (GLOBOCAN, 2020). Non-small-cell lung cancer (NSCLC) accounts for 85% to 90% of all cases, with a 5-year survival rate of approximately 18% across all stages of the disease, and only 3.5% for metastatic disease (Jemal et al., 2011) (Kantar, 2021; SEER, 2018). In the 1L setting, treatment typically consists of platinum-based chemotherapy in combination with immunotherapy, or a targeted therapy, depending on molecular and biomarker analysis and the histology of the tumor (NCCN, 2021d). More recently, the advent of PD-1 and programmed death ligand 1 (PD-L1) inhibitors have improved outcomes for patients without driver mutations (approximately 62% of the non-squamous population and 77% of the squamous population (Kantar, 2021)). More treatment alternatives are needed for patients whose tumors do not harbor certain oncogenic mutations or do not express the biomarker for checkpoint inhibitor (CPI) options. Novel combinations with complementary approaches to enhance response may further address the unmet need in this population. For patients in the 2L setting, SOC is limited to platinum-based chemotherapy, a CPI monotherapy or docetaxel with or without ramucirumab depending on the previous therapy received. For patients in the third-line (3L) setting, chemotherapy monotherapy is the standard. Novel therapies are needed to limit toxicity and potentially enhance efficacy in this population (NCCN, 2021d).
In one embodiment, wherein the tumor or cancer is lung cancer, this tumor or cancer is a non-small cell lung cancer (NSCLC), such as a squamous or non-squamous NSCLC. The tumor or cancer may in particular be a metastatic cancer, such as metastatic NSCLC.
In one embodiment, wherein the tumor or cancer is lung cancer, in particular NSCLC, the tumor or cancer does not have an epidermal growth factor (EGFR)-sensitizing mutation and/or anaplastic lymphoma (ALK) translocation/ROS1 rearrangement. EGFR-sensitizing mutations are those mutations that are amenable to treatment with an approved tyrosine kinase inhibitor (TKI).
In one embodiment, wherein the tumor or cancer is lung cancer, in particular NSCLC, the tumor or cancer comprises cancer cells and PD-L1 is expressed in ≥1% of the cancer cells. Such expression may be determined by any means and method known to the skilled person, such as by immunohistochemistry (IHC), such as determined by a local SOC testing (preferably an FDA-approved test) or at a central laboratory.
In one embodiment, wherein the tumor or cancer is lung cancer, in particular NSCLC, the tumor or cancer comprises cancer cells and PD-L1 is expressed in 1% to 49% of the cancer cells. Such expression may be determined by any means and method known to the skilled person, such as by immunohistochemistry (IHC), such as determined by a local SOC testing (preferably an FDA-approved test) or at a central laboratory.
In one embodiment, wherein the tumor or cancer is lung cancer, in particular NSCLC, the tumor or cancer comprises cancer cells and PD-L1 is expressed in ≥50% of the cancer cells. Such expression may be determined by any means and method known to the skilled person, such as by immunohistochemistry (IHC), such as determined by a local SOC testing (preferably an FDA-approved test) or at a central laboratory.
In one embodiment, the subject has not received prior systemic treatment of metastatic disease i.e., the subject has not received any systemic treatment of metastatic disease prior to receiving treatment according to the invention. According to this embodiment, the tumor or cancer is preferably a lung cancer, such as NSCLC.
In one embodiment, the subject has not received prior treatment with a checkpoint inhibitor/an immune checkpoint (ICP) inhibitor, i.e., before the treatment according to the first aspect, the subject has not received treatment with ICP inhibitor. In further embodiments, the subject has not received prior treatment with a PD-1 inhibitor or a PD-L1 inhibitor, such as anti-PD-1 antibody or an anti-PD-L1 antibody. In these embodiments the tumor or cancer is preferably a lung cancer, such as NSCLC.
In a further embodiment, the subject has not received prior treatment with a 4-1BB (CD137) targeted agent, with an antitumor vaccine, or with autologous cell immunotherapy. In one embodiment, the subject has not received prior treatment with an anti-4-1BB (CD137) antibody. In these embodiments the tumor or cancer is preferably a lung cancer, such as NSCLC.
In other embodiments the tumor or cancer has relapsed and/or is refractory after treatment, such as systemic treatment with a checkpoint inhibitor.
The subject may have received at least one prior line of systemic therapy, such as systemic therapy comprising a PD-1 inhibitor or a PD-L1 inhibitor, such as an anti-PD-1 antibody or an anti-PD-L1 antibody. The cancer or tumor may in particular have relapsed and/or is refractory, or the subject has progressed after treatment with a PD-1 inhibitor or a PD-L1 inhibitor, such as an anti PD-1 antibody or an anti-PD-L1 antibody, the PD-1 inhibitor or PD-L1 inhibitor being administered as monotherapy or as part of a combination therapy.
In particular embodiments the treatment according to the invention is provided to a subject having received prior treatment; e.g. as defined above, wherein the last prior treatment was with a PD1 inhibitor or PD-L1 inhibitor, such as an anti PD-1 antibody or an anti-PD-L1 antibody, the PD-1 inhibitor or PD-L1 inhibitor being administered as monotherapy or as part of a combination therapy. The last prior treatment may be with a PD1 inhibitor or PD-L1 inhibitor defined above.
Preferably, the therapy according to the invention is provided to a subject when the time from progression of that subject on last treatment with a PD1 inhibitor or PD-L1 inhibitor, such as an anti PD-1 antibody or an anti-PD-L1 antibody is 8 months or less, such as 7 months or less, 6 months or less, 5 months or less, 4 months or less, 3 months or less, 2 months or less, 1 month or less, 3 weeks or less or such as 2 weeks or less.
By analogy, it may be preferred to offer therapy according to the present invention to a subjects when the time from last dosing of a PD1 inhibitor or PD-L1 inhibitor, such as an anti PD-1 antibody or an anti-PD-L1 antibody as part of last prior treatment is 8 months or less, such as 7 months or less, 6 months or less, 5 months or less, 4 months or less, 3 months or less, 2 months or less, 1 month or less, 3 weeks or less or such as 2 weeks or less.
In further embodiments the cancer or tumor has relapsed and/or is refractory, or the subject has progressed during or after
Also, in these embodiments the tumor or cancer is preferably a lung cancer, such as NSCLC.
The subject receiving treatment according to the invention may in particular be a subject who has not received prior treatment with a taxane chemotherapeutic; e.g., docetaxel or paclitaxel, such as prior treatment of NSCLC with a taxane chemotherapeutic e.g., docetaxel.
The binding agent and the PD-1 inhibitor can be administered by any suitable way, such as intravenously, intraarterially, subcutaneously, intradermally, intramuscularly, intranodally, or intratumorally.
In one embodiment of the first aspect, the binding agent defined above is administered to the subject by systemic administration. Preferably, the binding agent is administered to the subject by intravenous injection or infusion. In one embodiment, the binding agent is administered in at least one treatment cycle.
In one embodiment, the antibody binding to PD-1 or antigen-binding fragment thereof is in particular administered to the subject by systemic administration. Preferably, the antibody binding to PD-1 or antigen-binding fragment thereof is administered to the subject by intravenous injection or infusion. In one embodiment, the antibody binding to PD-1 or antigen-binding fragment thereof is administered in at least one treatment cycle.
In one embodiment, the binding agent defined above and the antibody binding to PD-1 or antigen-binding fragment thereof are in particular administered to the subject by systemic administration. Preferably, the binding agent and the antibody binding to PD-1 or antigen-binding fragment thereof are administered to the subject by intravenous injection or infusion. In one embodiment, the binding agent and the antibody binding to PD-1 or antigen-binding fragment thereof are administered in at least one treatment cycle.
In one embodiment, each treatment cycle is about two weeks (14 days), three weeks (21 days) or four weeks (28 days), five weeks (35 days) or 6 weeks (48 days). In preferred embodiments each treatment cycle is three weeks (21 days). In other preferred embodiments, each treatment cycle is 6 weeks (48 days).
In particular embodiments, one dose of the binding agent defined above and one dose of the antibody binding to PD-1 or antigen-binding fragment thereof are administered or infused every second week (1Q2W), every third week (1Q3W) or every fourth week (1Q4W), every fifth week (1Q5W), preferably every third week (1Q3W). In other embodiments, one dose of the binding agent defined above and one dose of the antibody binding to PD-1 or antigen-binding fragment thereof are administered every six weeks (1Q6W). The amount of binding agent and the amount of antibody binding to PD-1 or antigen-binding fragment thereof are preferably as defined above.
In some embodiments, one dose or each dose is administered or infused on day 1 of each treatment cycle. For example, one dose of the binding agent defined above and one dose of the antibody binding to PD-1 or antigen-binding fragment thereof may be administered on day 1 of each treatment cycle.
In some embodiments a 100 mg dose of the binding agent defined above and a 200 mg dose of the antibody binding to PD-1 or antigen-binding fragment thereof are administered every three weeks (1Q3W).
In other embodiments a 100 mg dose of the binding agent defined above and a 400 mg dose of the antibody binding to PD-1 or antigen-binding fragment thereof are administered every six weeks (1Q6W).
In particular embodiments, 100 mg dose of the binding agent, which is acasunlimab or a biosimilar thereof and a 200 mg dose of the antibody binding to PD-1 or antigen-binding fragment thereof, which is pembrolizumab or a biosimilar thereof, are administered every three weeks (1Q3W), such as on day one of each three-week treatment cycle.
In particular embodiments, the tumor or cancer is NSCLC; and a 100 mg dose of the binding agent, which is acasunlimab or a biosimilar thereof and a 200 mg dose of the antibody binding to PD-1 or antigen-binding fragment thereof, which is pembrolizumab or a biosimilar thereof, are administered every three weeks (1Q3W), such as on day one of each three-week treatment cycle.
In other embodiments a 100 mg dose of the binding agent, which is acasunlimab or a biosimilar thereof and a 400 mg dose of the antibody binding to PD-1 or antigen-binding fragment thereof, which is pembrolizumab or a biosimilar thereof, are administered every six weeks (1Q6W), such as on day one of every six-week treatment cycle.
In still other embodiments, the tumor or cancer is NSCLC; and wherein a 100 mg dose of the binding agent, which is acasunlimab or a biosimilar thereof and a 400 mg dose of the antibody binding to PD-1 or antigen-binding fragment thereof, which is pembrolizumab, are administered every six weeks (1Q6W), such as on day one of every six-week treatment cycle.
The antibody binding to PD-1 or antigen-binding fragment thereof may be administered first, followed by the binding agent. Alternatively, the binding agent is administered first, followed by the antibody binding to PD-1 or antigen-binding fragment thereof.
Each dose may be administered or infused over a minimum of 30 minutes, such as over a minimum of 60 minutes, a minimum of 90 minutes, a minimum of 120 minutes or a minimum of 240 minutes.
The binding agent may in particular be administered by using intravenous (IV) infusion over 30 minutes, such as over a minimum of 40 minutes, a minimum of 50 minutes or such as over a minimum of 60 minutes.
The antibody binding to PD-1 or antigen-binding fragment thereof may in particular be administered as an intravenous infusion over 30 minutes, such as over a minimum of 40 minutes, a minimum of 50 minutes or such as over a minimum of 60 minutes.
The binding agent defined above and the antibody binding to PD-1 or antigen-binding fragment thereof may be administered simultaneously. In an alternative preferred embodiment, the binding agent and the PD-1 inhibitor are administered separately.
The binding agent defined above and the antibody binding to PD-1 or antigen-binding fragment thereof may be administered in any suitable form (e.g., naked as such). However, it is preferred that the binding agent and the PD-1 inhibitor, are administered in the form of any suitable pharmaceutical composition as described herein. In one embodiment, at least the binding agent and the antibody binding to PD-1 or antigen-binding fragment thereof are administered in the form of separate pharmaceutical compositions (i.e., one pharmaceutical composition for the binding agent and one pharmaceutical composition for the antibody binding to PD-1 or antigen-binding fragment thereof), preferably the binding agent and the antibody binding to PD-1 or antigen-binding fragment thereof are administered in the form of separate pharmaceutical compositions (i.e., one pharmaceutical composition for the binding agent and one pharmaceutical composition for the antibody binding to PD-1 or antigen-binding fragment thereof.
A composition or pharmaceutical composition may be formulated with a carrier, excipient and/or diluent as well as any other components suitable for pharmaceutical compositions, including known adjuvants, in accordance with conventional techniques such as those disclosed in Remington: The Science and Practice of Pharmacy, 19th Edition, Gennaro, Ed., Mack Publishing Co., Easton, PA, 1995. The pharmaceutically acceptable carriers or diluents as well as any known adjuvants and excipients should be suitable for the binding agent and/or the antibody binding to PD-1 or antigen-binding fragment and the chosen mode of administration. Suitability for carriers and other components of pharmaceutical compositions is determined based on the lack of significant negative impact on the desired biological properties of the chosen compound or pharmaceutical composition (e.g., less than a substantial impact [10% or less relative inhibition, 5% or less relative inhibition, etc.] upon antigen binding).
A composition, in particular the pharmaceutical composition of the binding agent defined above, the pharmaceutical composition of the antibody binding to PD-1 or antigen-binding fragment thereof, may include diluents, fillers, salts, buffers, detergents (e.g., a nonionic detergent, such as Tween-20 or Tween-80), stabilizers (e.g., sugars or protein-free amino acids), preservatives, solubilizers, and/or other materials suitable for inclusion in a pharmaceutical composition.
Pharmaceutically acceptable carriers, excipients or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R Gennaro edit. 1985).
Pharmaceutical carriers, excipients or diluents can be selected with regards to the intended route of administration and standard pharmaceutical practice.
Pharmaceutically acceptable carriers include any and all suitable solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonicity agents, antioxidants and absorption-delaying agents, and the like that are physiologically compatible with the active compound, in particular a binding agent defined above and the antibody binding to PD-1 or antigen-binding fragment thereof.
Examples of suitable aqueous and non-aqueous carriers which may be employed in the (pharmaceutical) compositions include water, saline, phosphate buffered saline, ethanol, dextrose, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, corn oil, peanut oil, cottonseed oil, and sesame oil, carboxymethyl cellulose colloidal solutions, tragacanth gum and injectable organic esters, such as ethyl oleate, and/or various buffers. Other carriers are well known in the pharmaceutical arts.
Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the (pharmaceutical) compositions is contemplated.
The term “excipient” as used herein refers to a substance which may be present in a (pharmaceutical) composition of the present disclosure but is not an active ingredient. Examples of excipients, include without limitation, carriers, binders, diluents, lubricants, thickeners, surface active agents, preservatives, stabilizers, emulsifiers, buffers, flavoring agents, or colorants.
The term “diluent” relates a diluting and/or thinning agent. Moreover, the term “diluent” includes any one or more of fluid, liquid or solid suspension and/or mixing media. Examples of suitable diluents include ethanol, glycerol and water
A (pharmaceutical) composition may also comprise pharmaceutically acceptable antioxidants for instance (1) water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal-chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
A (pharmaceutical) composition may also comprise isotonicity agents, such as sugars, polyalcohols, such as mannitol, sorbitol, glycerol or sodium chloride in the composition.
A (pharmaceutical) composition may also contain one or more adjuvants appropriate for the chosen route of administration such as preservatives, wetting agents, emulsifying agents, dispersing agents, preservatives or buffers, which may enhance the shelf life or effectiveness of the composition. The composition as used herein may be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and micro-encapsulated delivery systems. Such carriers may include gelatin, glyceryl monostearate, glyceryl distearate, biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, poly-ortho esters, and polylactic acid alone or with a wax, or other materials well known in the art. Methods for the preparation of such formulations are generally known to those skilled in the art, see e.g. Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.
“Pharmaceutically acceptable salts” comprise, for example, acid addition salts which may, for example, be formed by using a pharmaceutically acceptable acid such as hydrochloric acid, sulfuric acid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoic acid, citric acid, tartaric acid, carbonic acid or phosphoric acid. Furthermore, suitable pharmaceutically acceptable salts may include alkali metal salts (e.g., sodium or potassium salts); alkaline earth metal salts (e.g., calcium or magnesium salts); ammonium (NH4+); and salts formed with suitable organic ligands (e.g., quaternary ammonium and amine cations formed using counteranions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, alkyl sulfonate and aryl sulfonate). Illustrative examples of pharmaceutically acceptable salts include, but are not limited to, acetate, adipate, alginate, arginate, ascorbate, aspartate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, butyrate, calcium edetate, camphorate, camphorsulfonate, camsylate, carbonate, chloride, citrate, clavulanate, cyclopentanepropionate, digluconate, dihydrochloride, dodecylsulfate, edetate, edisylate, estolate, esylate, ethanesulfonate, formate, fumarate, galactate, galacturonate, gluceptate, glucoheptonate, gluconate, glutamate, glycerophosphate, glycolylarsanilate, hemisulfate, heptanoate, hexanoate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, hydroxynaphthoate, iodide, isobutyrate, isothionate, lactate, lactobionate, laurate, lauryl sulfate, malate, maleate, malonate, mandelate, mesylate, methanesulfonate, methylsulfate, mucate, 2-naphthalenesulfonate, napsylate, nicotinate, nitrate, N-methylglucamine ammonium salt, oleate, oxalate, pamoate (embonate), palmitate, pantothenate, pectinate, persulfate, 3-phenylpropionate, phosphate/diphosphate, phthalate, picrate, pivalate, polygalacturonate, propionate, salicylate, stearate, sulfate, suberate, succinate, tannate, tartrate, teoclate, tosylate, triethiodide, undecanoate, valerate, and the like (see, for example, S. M. Berge et al., “Pharmaceutical Salts”, J. Pharm. Sci., 66, pp. 1-19 (1977)). Salts which are not pharmaceutically acceptable may be used for preparing pharmaceutically acceptable salts and are included in the present disclosure.
In one embodiment, the binding agent, and the PD-1 inhibitor, used herein may be formulated to ensure proper distribution in vivo. Pharmaceutically acceptable carriers for parenteral administration include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art. Except in so far as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Other active or therapeutic compounds may also be incorporated into the compositions.
Pharmaceutical compositions for injection must typically be sterile and stable under the conditions of manufacture and storage. The composition may be formulated as a solution, micro-emulsion, liposome, or other ordered structure suitable to high drug concentration. The carrier may be an aqueous or a non-aqueous solvent or dispersion medium containing for instance water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. The proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as glycerol, mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions may be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin. Sterile injectable solutions may be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients e.g. as enumerated above, as required, followed by sterilization microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients e.g. from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, examples of methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
Sterile injectable solutions may be prepared by incorporating the active compounds in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, examples of methods of preparation are vacuum-drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
In certain embodiments the binding agent for use according to the invention is formulated in a composition or formulation comprising histidine, sucrose and Polysorbate-80, and having a pH from about 5 to about 6, such as from 5 to 6. In particular, the binding agent for use according to the invention may be in a composition or formulation comprising about 20 mM histidine, about 250 mM Sucrose, about 0.02% Polysorbate-80, and having a pH of about 5.5, such as a composition or formulation comprising 20 mM histidine, 250 mM Sucrose, 0.02% Polysorbate-80, and having a pH of 5.5. The formulation may in particular embodiments comprise about 10 to about 30 mg binding agent/mL, such as 10-30 mg binding agent/mL, in particular about 20 mg binding agent/mL, such as 20 mg binding agent/mL.
The binding agent for use according to the invention may be provided in a composition as defined above and may then be diluted in 0.9% NaCl (saline) prior to administration.
In a second aspect, the present disclosure provides a kit comprising
The embodiments disclosed herein with respect to the first aspect (in particular regarding the binding agent, and the antibody binding to PD-1, or an antigen-binding fragment thereof also apply to the kit of the second aspect. In one embodiment, the kit comprises at least two containers, wherein one thereof contains the binding agent (as such or in the form of a (pharmaceutical) composition) and the second container contains the antibody binding to PD-1, or an antigen-binding fragment thereof (as such or in the form of a (pharmaceutical) composition).
In a third aspect, the present disclosure provides a kit of the second aspect for use in a method for reducing or preventing progression of a tumor or treating cancer in a subject. The embodiments disclosed herein with respect to the first aspect (in particular regarding the binding agent, the PD-1 inhibitor, the treatment regimen, the specific tumor/cancer, and the subject) and/or the second aspect also apply to the kit for use of the third aspect.
In a fourth aspect, the present disclosure provides a method for reducing or preventing progression of a tumor or treating cancer in a subject, said method comprising administering to said subject a binding agent prior to, simultaneously with, or after administration of an antibody binding to PD-1, or an antigen-binding fragment thereof, wherein the binding agent comprises a first binding region binding to CD137 and a second binding region binding to PD-L1;
The embodiments disclosed herein with respect to the first aspect (in particular regarding the binding agent, the PD-1 inhibitor, the treatment regimen, the specific tumor/cancer, and the subject) also apply to the method of the fourth aspect.
In a further aspect, the present disclosure provides an antibody binding to PD-1, or an antigen-binding fragment thereof for use in a method for reducing or preventing progression of a tumor or treating cancer in a subject, said method comprising administering to said subject the PD-1 inhibitor prior to, simultaneously with, or after administration of an antibody binding to PD-1, or an antigen-binding fragment thereof,
wherein the antibody comprises a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 43, 44 and 45, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 46, 47 and 48, respectively, or the antibody comprises a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 62, 63 and 64, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 65, 66 and 67, respectively, and
wherein the binding agent comprises a first binding region binding to CD137 and a second binding region binding to PD-L1
The embodiments disclosed herein with respect to the first aspect (in particular regarding the binding agent, the PD-1 inhibitor, the optional one or more additional therapeutic agents, the treatment regimen, the specific tumor/cancer, and the subject) also apply to the PD-1 inhibitor for use of this further aspect.
Citation of documents and studies referenced herein is not intended as an admission that any of the foregoing is pertinent prior art. All statements as to the contents of these documents are based on the information available to the applicants and do not constitute any admission as to the correctness of the contents of these documents.
The description (including the following examples) is presented to enable a person of ordinary skill in the art to make and use the various embodiments. Descriptions of specific devices, techniques, and applications are provided only as examples. Various modifications to the examples described herein will be readily apparent to those of ordinary skill in the art, and the general principles defined herein may be applied to other examples and applications without departing from the spirit and scope of the various embodiments. Thus, the various embodiments are not intended to be limited to the examples described herein and shown, but are to be accorded the scope consistent with the claims.
1. A binding agent for use in a method for reducing or preventing progression of a tumor or treating cancer in a subject, said method comprising administering to said subject the binding agent prior to, simultaneously with, or after administration of an antibody binding to Programmed Death-1 (PD-1), or an antigen-binding fragment thereof,
2. The binding agent for use according to item 1, wherein the antibody binding to PD-1 or the antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence having at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 49 and a light chain variable region comprising an amino acid sequence having at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 50.
3. The binding agent for use of anyone of the preceding items, wherein the antibody binding to PD-1 or the antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 49 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 50.
4. The binding agent for use of any one of the preceding items, wherein the antibody binding to PD-1 or the antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 51 and a light chain comprising the amino acid sequence of SEQ ID NO: 52.
5. The binding agent for use of any one of the preceding items, wherein the antibody binding to PD-1 is pembrolizumab or a biosimilar thereof.
6. The binding agent for use of any one of the preceding items, wherein PD-L1 is human PD-L1, in particular human PD-L1 comprising the sequence set forth in SEQ ID NO: 40, and/or CD137 is human CD137, in particular human CD137 comprising the sequence set forth in SEQ ID NO: 38.
7. The binding agent for use of any one of the preceding items, wherein the first binding region of the binding agent comprises a heavy chain variable region (VH) comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to SEQ ID NO: 1 or 9 and a light chain variable region (VL) region and comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to SEQ ID NO: 5 or 10.
8. The binding agent for use of any one of the preceding items, wherein the second binding region of the binding agent comprises a heavy chain variable region (VH) comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 25 100% sequence identity to SEQ ID NO: 11 and a light chain variable region (VL) region comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to SEQ ID NO: 15.
9. The binding agent for use of any one of the preceding items, wherein the first binding region of the binding agent comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 1 or 9 and a light chain variable region (VL) region comprising the amino acid sequence set forth in SEQ ID NO: 5 or 10.
10. The binding agent for use of any one of the preceding items, wherein the second binding region of the binding agent comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 11 and a light chain variable region (VL) region comprising the amino acid sequence set forth in SEQ ID NO: 15.
11. The binding agent for use of any one of the preceding items, wherein
12. The binding agent for use of any one of the preceding items, wherein the binding agent is a multispecific antibody, such as a bispecific antibody.
13. The binding agent for use of any one of the preceding items, wherein the binding agent is in the format of a full-length antibody or an antibody fragment.
14. The binding agent for use of any one of the preceding items, wherein each variable region comprises three complementarity determining regions (CDR1, CDR2, and CDR3) and four framework regions (FR1, FR2, FR3, and FR4).
15. The binding agent for use of item 13, wherein said complementarity determining regions and said framework regions are arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
16. The binding agent for use of any one of the preceding items, wherein the binding agent comprises
17. The binding agent for use of any one of the preceding items, wherein the binding agent comprises
18. The binding agent for use of any one of the preceding items, wherein the binding agent is an antibody comprising a first binding arm and a second binding arm, wherein the first binding arm comprises
19. The binding agent for use of any one of the preceding items, wherein the binding agent comprises
20. The binding agent for use of any one of the preceding items, wherein said binding agent comprises
21. The binding agent for use of any one of items 16-20, wherein each of the first and second heavy chain constant regions (CH) comprises one or more of a constant heavy chain 1 (CH1) region, a hinge region, a constant heavy chain 2 (CH2) region and a constant heavy chain 3 (CH3) region, preferably at least a hinge region, a CH2 region and a CH3 region.
22. The binding agent for use of any one of items 16-21, wherein each of the first and second heavy chain constant regions (CHs) comprises a CH3 region and wherein the two CH3 regions comprise asymmetrical mutations.
23. The binding agent for use of any one of items 16-21, wherein in said first heavy chain constant region (CH) at least one of the amino acids in a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in a human IgG1 heavy chain according to EU numbering has been substituted, and in said second heavy chain constant region (CH) at least one of the amino acids in a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in a human IgG1 heavy chain according to EU numbering has been substituted, and wherein said first and said second heavy chains are not substituted in the same positions.
24. The binding agent for use of item 23, wherein (i) the amino acid in the position corresponding to F405 in a human IgG1 heavy chain according to EU numbering is L in said first heavy chain constant region (CH), and the amino acid in the position corresponding to K409 in a human IgG1 heavy chain according to EU numbering is R in said second heavy chain constant region (CH), or (ii) the amino acid in the position corresponding to K409 in a human IgG1 heavy chain according to EU numbering is R in said first heavy chain, and the amino acid in the position corresponding to F405 in a human IgG1 heavy chain according to EU numbering is L in said second heavy chain.
25. The binding agent for use of any of the preceding items, wherein said binding agent induces Fc-mediated effector function to a lesser extent compared to another antibody comprising the same first and second antigen binding regions and two heavy chain constant regions (CHs) comprising human IgG1 hinge, CH2 and CH3 regions.
26. The binding agent for use of item 25, wherein said first and second heavy chain constant regions (CHs) are modified so that the antibody induces Fc-mediated effector function to a lesser extent compared to an antibody which is identical except for comprising non-modified first and second heavy chain constant regions (CHs).
27. The binding agent for use of item 26, wherein each of said non-modified first and second heavy chain constant regions (CHs) comprises the amino acid sequence set forth in SEQ ID NO: 19 or 25.
28. The binding agent for use of item 26 or 27, wherein said Fc-mediated effector function is measured by binding to Fcγ receptors, binding to C1q, or induction of Fe-mediated crosslinking of Fcγ receptors.
29. The binding agent for use of item 28, wherein said Fc-mediated effector function is measured by binding to C1q.
30. The binding agent for use of any one of items 25-29, wherein said first and second heavy chain constant regions have been modified so that binding of C1q to said antibody is reduced compared to a wild-type antibody, preferably reduced by at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100%, wherein C1q binding is preferably determined by ELISA.
31. The binding agent for use of any one of the preceding items, wherein in at least one of said first and second heavy chain constant regions (CH), one or more amino acids in the positions corresponding to positions L234, L235, D265, N297, and P331 in a human IgG1 heavy chain according to EU numbering, are not L, L, D, N, and P, respectively.
32. The binding agent for use of item 31, wherein the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain according to EU numbering are F and E, respectively, in said first and second heavy chains.
33. The binding agent for use of item 31 or 32, wherein the positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain according to EU numbering are F, E, and A, respectively, in said first and second heavy chain constant regions (HCs).
34. The binding agent for use of any one of items 31-33, wherein the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain according to EU numbering of both the first and second heavy chain constant regions are F and E, respectively, and wherein (i) the position corresponding to F405 in a human IgG1 heavy chain according to EU numbering of the first heavy chain constant region is L, and the position corresponding to K409 in a human IgG1 heavy chain according to EU numbering of the second heavy chain is R, or (ii) the position corresponding to K409 in a human IgG1 heavy chain according to EU numbering of the first heavy chain constant region is R, and the position corresponding to F405 in a human IgG1 heavy chain according to EU numbering of the second heavy chain is L.
35. The binding agent for use of any one of items 31-34, wherein the positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain according to EU numbering of both the first and second heavy chain constant regions are F, E, and A, respectively, and wherein (i) the position corresponding to F405 in a human IgG1 heavy chain according to EU numbering of the first heavy chain constant region is L, and the position corresponding to K409 in a human IgG1 heavy chain according to EU numbering of the second heavy chain constant region is R, or (ii) the position corresponding to K409 in a human IgG1 heavy chain according to EU numbering of the first heavy chain is R, and the position corresponding to F405 in a human IgG1 heavy chain according to EU numbering of the second heavy chain is L.
36. The binding agent for use of any one of items 16-35, wherein the constant region of said first and/or second heavy chain comprises or consists essentially of or consists of an amino acid sequence selected from the group consisting of
37. The binding agent for use of any one of items 16-36, wherein the constant region of said first or second heavy chain, such as the second heavy chain, comprises or consists essentially of or consists of an amino acid sequence selected from the group consisting of
38. The binding agent for use of any one of items 16-36, wherein the constant region of said first or second heavy chain, such as the first heavy chain comprises or consists essentially of or consists of an amino acid sequence selected from the group consisting of
39. The binding agent for use of any one of items 16-15, wherein the constant region of said first and/or second heavy chain comprises or consists essentially of or consists of an amino acid sequence selected from the group consisting of
40. The binding agent for use of any one of items 16-39, wherein the constant region of said first and/or second heavy chain, such as the second heavy chain, comprises or consists essentially of or consists of an amino acid sequence selected from the group consisting of
41. The binding agent for use of any one of items 16-40, wherein the constant region of said first and/or second heavy chain, such as the first heavy chain, comprises or consists essentially of or consists of an amino acid sequence selected from the group consisting of
42. The binding agent for use of any one of the preceding items, wherein said binding agent comprises a kappa (κ) light chain constant region.
43. The binding agent for use of any one of the preceding items, wherein said binding agent comprises a lambda (λ) light chain constant region.
44. The binding agent for use of any one of the preceding items, wherein said first light chain constant region is a kappa (κ) light chain constant region or a lambda (λ) light chain constant region.
45. The binding agent for use of any one of the preceding items, wherein said second light chain constant region is a lambda (λ) light chain constant region or a kappa (κ) light chain constant region.
46. The binding agent for use of any one of the preceding items, wherein said first light chain constant region is a kappa (κ) light chain constant region and said second light chain constant region is a lambda (λ) light chain constant region or said first light chain constant region is a lambda (λ) light chain constant region and said second light chain constant region is a kappa (κ) light chain constant region.
47. The binding agent for use of any one of items 42-46, wherein the kappa (κ) light chain comprises an amino acid sequence selected from the group consisting of
48. The binding agent for use of any one of items 43-47, wherein the lambda (λ) light chain comprises an amino acid sequence selected from the group consisting of
49. The binding agent for use of any one of the preceding items, wherein the binding agent is of an isotype selected from the group consisting of IgG1, IgG2, IgG3, and IgG4.
50. The binding agent for use of any one of the preceding items, wherein the binding agent is a full-length IgG1 antibody.
51. The binding agent for use of any one of the preceding items, wherein the binding agent is an antibody of the IgG1m(f) allotype.
52. The binding agent for use of any one of the preceding items, wherein the binding agent comprises
53. The binding agent for use according to any one of the preceding items, wherein the binding agent is acasunlimab or a biosimilar thereof.
54. The binding agent for use according to any one of the preceding items, wherein the binding agent is in a composition or formulation comprising histidine, sucrose and Polysorbate-80, and has a pH from 5 to 6.
55. The binding agent for use according to any one of the preceding items, wherein the binding agent is in a composition or formulation comprising about 20 mM histidine, about 250 mM Sucrose, about 0.02% Polysorbate-80, and having a pH of about 5.5.
56. The binding agent for use according to any one of the preceding items, wherein the binding agent is in a composition or formulation comprising 10-30 mg binding agent/mL, such as 20 mg binding agent/mL.
57. The binding agent for use according to any one of the preceding items, wherein the binding agent is in a composition as defined in any one of items 54 to 56 and is diluted in 0.9% NaCl (saline) prior to administration.
58. The binding agent for use of any one of the preceding items, wherein the subject is a human subject.
59. The binding agent for use of any one of the preceding items, wherein the tumor or cancer is a solid tumor or cancer.
60. The binding agent for use according to any one of the preceding items, wherein said tumor is a PD-L1 positive tumor.
61. The binding agent for use of any one of the preceding items, wherein the tumor or cancer is selected from the group consisting of melanoma, ovarian cancer, lung cancer (e.g., non-small cell lung cancer (NSCLC)), colorectal cancer, head and neck cancer, gastric cancer, breast cancer, renal cancer, urothelial cancer, bladder cancer, esophageal cancer, pancreatic cancer, hepatic cancer, thymoma and thymic carcinoma, brain cancer, glioma, adrenocortical carcinoma, thyroid cancer, other skin cancers, sarcoma, multiple myeloma, leukemia, lymphoma, myelodysplastic syndromes, endometrial cancer, prostate cancer, penile cancer, cervical cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, Merkel cell carcinoma and mesothelioma.
62. The binding agent for use according to any one of the preceding items, wherein the tumor or cancer is selected from the group consisting of lung cancer (e.g. non-small cell lung cancer (NSCLC), urothelial cancer (cancer of the bladder, ureter, urethra, or renal pelvis), endometrial cancer (EC), breast cancer (e.g. triple negative breast cancer (TNBC)) and squamous cell carcinoma of the head and neck (SCCHN) (e.g. cancer of the oral cavity, pharynx or larynx).
63. The binding agent for use of item 61 or 62, wherein the tumor or cancer is lung cancer, in particular a non-small cell lung cancer (NSCLC), such as a squamous or non-squamous NSCLC.
64. The binding agent for use of any one of items 61 to 63, wherein the tumor or cancer is metastatic, such as metastatic NSCLC.
65. The binding agent for use of item 61 to 64, wherein the lung cancer, in particular NSCLC, does not have an epidermal growth factor (EGFR)-sensitizing mutation and/or anaplastic lymphoma (ALK) translocation/ROS1 rearrangement.
66. The binding agent for use of any one of items 61 to 65, wherein the lung cancer, in particular NSCLC, comprises cancer cells and PD-L1 is expressed in ≥1% of the cancer cells or tumor cells e.g. as assessed by immunohistochemistry (IHC).
67. The binding agent for use of item 66, wherein the lung cancer, in particular NSCLC, comprises cancer cells and PD-L1 is expressed in 1% to 49% of the cancer cells or tumor cells e.g. as assessed by immunohistochemistry (IHC).
68. The binding agent for use of item 66, wherein the lung cancer, in particular NSCLC, comprises cancer cells and PD-L1 is expressed in ≥50% of the cancer cells or tumor cells e.g. as assessed by immunohistochemistry (IHC).
69. The binding agent for use of the preceding items, wherein the subject has not received prior systemic treatment of metastatic disease.
70. The binding agent for use of any one of the preceding items, wherein the subject has not received prior treatment with a checkpoint inhibitor; e.g., a PD-1 inhibitor or a PD-L1 inhibitor, such as an anti-PD-1 antibody or an anti-PD-L1 antibody.
71. The binding agent for use of any one of the preceding items, wherein the subject has not received prior treatment with a 4-1BB (CD137) targeted agent, such as an anti-4-1BB (CD137) antibody, with an antitumor vaccine, or with autologous cell immunotherapy.
72. The binding agent for use of any one of items 1 to 68, wherein the tumor or cancer has relapsed and/or is refractory after treatment, such as systemic treatment with a checkpoint inhibitor.
73. The binding agent for use of any one of items 1 to 68 and 72, wherein the subject has received at least 1 prior line of systemic therapy, such as systemic therapy comprising a PD-1 inhibitor or a PD-L1 inhibitor, such as an anti-PD-1 antibody or an anti-PD-L1 antibody.
74. The binding agent for use of any one of items 1 to 68, 72 and 73, wherein the cancer or tumor has relapsed and/or is refractory, or the subject has progressed after treatment with a PD-1 inhibitor or a PD-L1 inhibitor, such as an anti PD-1 antibody or an anti-PD-L1 antibody, the PD-1 inhibitor or PD-L1 inhibitor being administered as monotherapy or as part of a combination therapy.
75. The binding agent for use of any one of items 1 to 68 and 72 to 74, wherein last prior treatment was with a PD1 inhibitor or PD-L1 inhibitor, such as an anti PD-1 antibody or an anti-PD-L1 antibody, the PD-1 inhibitor or PD-L1 inhibitor being administered as monotherapy or as part of a combination therapy.
76. The binding agent for use of any one of items 1 to 68 and 72 to 74, wherein the time from progression on last treatment with a PD1 inhibitor or PD-L1 inhibitor, such as an anti PD-1 antibody or an anti-PD-L1 antibody is 8 months or less, such as 7 months or less, 6 months or less, 5 months or less, 4 months or less, 3 months or less, 2 months or less, 1 month or less, 3 weeks or less or such as 2 weeks or less.
77. The binding agent for use of any one of items 1 to 68 and 72 to 74, wherein the time from last dosing of a PD1 inhibitor or PD-L1 inhibitor, such as an anti PD-1 antibody or an anti-PD-L1 antibody as part of last prior treatment is 8 months or less, such as 7 months or less, 6 months or less, 5 months or less, 4 months or less, 3 months or less, 2 months or less, 1 month or less, 3 weeks or less or such as 2 weeks or less.
78. The binding agent for use of any one of items 1 to 68 and 72 to 74, wherein the cancer or tumor has relapsed and/or is refractory, or the subject has progressed during or after
79. The binding agent for use of any one of the preceding items, wherein the subject has not received prior treatment with a taxane chemotherapeutic agent e.g., docetaxel, such as prior treatment of NSCLC with a taxane chemotherapeutic agent e.g., docetaxel.
80. The binding agent for use of any one of the preceding items, wherein the binding agent and the antibody binding to PD-1, or the antigen-binding fragment thereof are administered in at least one treatment cycle, each treatment cycle being three weeks (21 days) or six weeks (42 days).
81. The binding agent for use of any one of the preceding items, wherein one dose of the binding agent and one dose of the antibody binding to PD-1, or the antigen-binding fragment thereof are administered every third week (1Q3W).
82. The binding agent for use of any one of the preceding items, wherein one dose of the binding agent and one dose of the antibody binding to PD-1, or the antigen-binding fragment thereof are administered every six weeks (1Q6W).
83. The binding agent for use of any one of the preceding items, wherein one dose of the binding agent and one dose of the antibody binding to PD-1, or the antigen-binding fragment thereof are administered on day 1 of each treatment cycle.
84. The binding agent for use of any one of the preceding items, wherein the amount of said binding agent administered in each dose and/or in each treatment cycle is 100 mg.
85. The binding agent for use of any one of the preceding items, wherein the amount of said antibody binding to PD-1, or the antigen-binding fragment thereof administered in each dose and/or in each treatment cycle is 200 mg.
86. The binding agent for use of any one of the preceding items, wherein the amount of said antibody binding to PD-1, or the antigen-binding fragment thereof administered in each dose and/or in each treatment cycle is 400 mg.
87. The binding agent for use of any one of the preceding items, wherein a 100 mg dose of the binding agent and a 200 mg dose of the antibody binding to PD-1, or the antigen-binding fragment thereof are administered every three weeks (1Q3W).
88. The binding agent for use of any one of the preceding items, wherein a 100 mg dose of the binding agent and a 400 mg dose of the antibody binding to PD-1, or the antigen-binding fragment thereof are administered every six weeks (1Q6W).
89. The binding agent for use of any one of the preceding items, wherein the tumor or cancer is NSCLC; and wherein a 100 mg dose of the binding agent, which is acasunlimab or a biosimilar thereof and a 200 mg dose of the antibody binding to PD-1, which is pembolizumab, are administered every three weeks (1Q3W), such as on day one of each three-week treatment cycle.
90. The binding agent for use of any one of items 1-88, wherein the tumor or cancer is NSCLC; and wherein a 100 mg dose of the binding agent, which is acasunlimab or a biosimilar thereof and a 400 mg dose of the antibody binding to PD-1, which is pembolizumab, are administered every six weeks (1Q6W), such as on day one of every six-week treatment cycle.
91. The binding agent for use of any one of the preceding items, wherein the antibody binding to PD-1, or the antigen-binding fragment thereof is administered first, followed by the binding agent.
92. The binding agent for use of any one of the preceding items, wherein the binding agent is administered by using intravenous (IV) infusion over a minimum of 30 minutes, such as over a minimum of 60 minutes.
93. The binding agent for use of any one of the preceding items, wherein the binding agent is administered by using intravenous (IV) infusion over 30 minutes.
94. The binding agent for use of any one of the preceding items, wherein the PD-1 inhibitor is administered as an intravenous infusion over 30 minutes.
95. A kit comprising
96. The kit according to item 95, wherein the binding agent and/or the antibody binding to PD-1, or the antigen-binding fragment thereof is as defined in any one of items 1 to 94.
97. The kit according to item 95 or 96, wherein the binding agent, and the antibody binding to PD-1, or the antigen-binding fragment thereof are for systemic administration, in particular for injection or infusion, such as intravenous injection or infusion.
98. The kit according to any one of items 95-97 for use in a method for reducing or preventing progression of a tumor or treating cancer in a subject.
99. The kit for use according to item 98, wherein the tumor or cancer and/or the subject and/or the method is/are as defined in any one of items 1-94.
100. A method for reducing or preventing progression of a tumor or treating cancer in a subject, said method comprising administering to said subject a binding agent prior to, simultaneously with, or after administration of an antibody binding to PD-1, or an antigen-binding fragment thereof, wherein the binding agent comprises a first binding region binding to CD137 and a second binding region binding to PD-L1
101. The method of item 100, wherein the tumor or cancer and/or the subject and/or the method and/or the binding agent and/or the PD-1 inhibitor is/are as defined in any one of items 1-94.
Further aspects of the present disclosure are disclosed herein.
Monocyte and T Cells from Healthy Donors
CD14+ monocytes and purified CD8+ T cells were obtained from Precision Medicine or BioIVT. Allogeneic donor pairs were used for the allogenic mixed lymphocyte reaction (MLR assay).
Human CD14+ monocytes were obtained from healthy donors (see above). For differentiation into immature dendritic cells (iDCs), 1-1.5×106 monocytes/mL were cultured for six days in Roswell Park Memorial Institute (RPMI) 1640 complete medium (ATCC modification formula; ThermoFisher, cat. no. A1049101) supplemented with 10% heat-inactivated Fetal Bovine Serum (FBS; Gibco, cat. no. 16140071), 100 ng/mL granulocyte-macrophage colony-stimulating factor (GM-CSF; BioLegend, cat. no. 766106) and 300 ng/mL interleukin-4 (IL-4; BioLegend, cat. no. 766206) in T25 culture flasks (Falcon, cat. no. 353108) at 37° C. Once during these six days, the medium was replaced with fresh medium with supplements.
Maturation of iDCs
To mature the iDCs, the cells were harvested by collecting non-adherent cells, counted, incubated at 1-1.5×106 cells/mL in RPMI 1640 complete medium supplemented with 10% FBS, 100 ng/mL GM-CSF, 300 ng/mL IL-4 and 1× with lipopolysaccharide (LPS; ThermoFisher, cat. no. 00-4976-93) for 24 h prior to start of the MLR assay at 37° C.
One day prior to the start of an MLR assay, purified CD8+ T cells, obtained from allogeneic healthy donors, were thawed. Cells were resuspended at 1×106 cells/mL in RPMI 1640 complete medium supplemented with 10% FBS and 10 ng/mL IL-2 (BioLegend, cat. no. 589106) and incubated O/N at 37° C.
The next day, the LPS-matured dendritic cells (mDCs, see Maturation of iDCs) and allogeneic purified CD8+ T cells were harvested and resuspended in AIM-V medium (ThermoFisher, cat. no. 12055091) at 4×105 cells/mL and 4×106 cells/mL, respectively.
In co-cultures, 20,000 mDCs were incubated with 200,000 allogeneic purified CD8+ T cells (DC:T cell ratio of 1:10) in the presence of GEN1046 (0.001-30 μg/mL) either alone or in combination with research-grade pembrolizumab (0.1-30 μg/mL or 0.1-100 μg/mL), research-grade pembrolizumab (0.1-30 μg/mL or 0.1-100 μg/mL), bsIgG1-PD-L1×ctrl (30 μg/mL), bsIgG1-ctrl×4-1BB (30 μg/mL), isotype control antibodies IgG4 (100 μg/mL), or IgG1-ctrl-FEAL (30 μg/mL; Table 5) in AIM-V medium in a 96-well round-bottom plate (Falcon, cat. no. 353227) at 37° C. After 5 days, the plates were centrifuged at 500×g for 5 min and the supernatant was carefully transferred from each well to a new 96-well round bottom plate.
The collected supernatants from the MLR assay were analyzed for interferon (IFN)γ levels by enzyme-linked immunosorbent assay (ELISA) using an Alpha Lisa IFNγ kit (Perkin Elmer, cat. no. AL217) on an Envision instrument, according to the manufacturer's instructions. TNFα and IL-2 were measured as part of the Milliplex MAP-Human cytokine/TH17 panel (Millipore Sigma, cat. no. SPR1526) on a Luminex FLEXMAP 3D instrument.
1Control binding moiety based on anti-HIV gp120 antibody IgG1-b12 (Barbas et al., J Mol Biol 230: 812-823)
GEN1046 induced IL-2 secretion in co-cultures of purified CD8+ T cells and allogeneic mDCs compared to IgG1-ctrl-FEAL in three donor pairs (see
In addition, GEN1046 induced IFNγ secretion in co-cultures of purified CD8+ T cells and allogeneic mDCs compared to IgG1-ctrl-FEAL in three donor pairs (see
Furthermore, GEN1046 induced TNFα secretion in co-cultures of purified CD8+ T cells and allogeneic mDCs compared to IgG1-ctrl-FEAL in three donor pairs (see
Together, these results indicate that combining GEN1046 with pembrolizumab potentiates IFNγ, IL-2 and TNFα secretion relative to each antibody alone in an mDC/CD8+ T cell MLR assay. While potentiation of IFNγ was observed mainly at low concentrations of GEN1046, combination of GEN1046 with pembrolizumab showed potentiation of IL-2 at multiple concentrations.
MC38 mouse colon cancer cells were cultured in Dulbecco's Modified Eagle Medium supplemented with 10% heat-inactivated fetal bovine serum at 37° C., 5% CO2. MC38 cells were harvested from a cell culture growing in log-phase and quantified.
MC38 cells (1×106 tumor cells in 100 μL PBS) were injected subcutaneously in the right lower flank of female C57BL/6 mice (obtained from Vital River Laboratories Research Models and Services; age 6-8 weeks at start of experiment).
Tumor growth was evaluated three times per week using a caliper. Tumor volumes (mm3) were calculated from caliper measurements as ([length]×[width]2)/2, where the length is the longest tumor dimension and the width is the longest tumor dimension perpendicular to the length.
Treatment was initiated when tumors had reached a median volume of 64 mm3. Mice were randomized into groups (n=10/group) with equal average tumor volume prior to treatment (64 mm3). On treatment days, the mice were injected intraperitoneally with mbsIgG2a-PD-L1×4-1BB (5 mg/kg; injection volume of 10 μL/g body weight; two doses weekly for three weeks [2QW×3]), an anti-mouse PD-1 antibody (anti-mPD-1; 10 mg/kg; injection volume of 10 μL/g body weight; 2QW×3; clone RMP1-14; Leinco Technologies, cat. no. P372), a combination of mbsIgG2a-PD-L1×4-1BB (5 mg/kg) with anti-mPD-1 (10 mg/kg; in two separate injections [mbsIgG2a-PD-L1×4-1BB followed by anti-mPD-1 after 20 min] with an injection volume of 10 μL/g body weight; 2QW×3), or PBS with an injection volume of 10 μL/g body weight (Table 6).
The mice were monitored daily for clinical signs of illness. Body weight measurements were performed three times a week after randomization. The experiment ended for the individual mice when the tumor volume exceeded 1500 mm3 or when the animals reached humane endpoints (e.g. when mice showed body weight loss >20%, when tumors showed ulceration [>75%], when serious clinical signs were observed and/or when the tumor growth blocked the physical activity of the mouse).
a2QW × 3: two doses weekly for three weeks
Rapid tumor outgrowth was observed in MC38-bearing mice treated with PBS (
These results provide rationale for evaluating the combination of GEN1046 with an anti-PD-1 antibody to further amplify the anti-tumor immune response in cancer patients to produce durable and deep clinical responses and enhance survival.
1Tumor volume <500 mm3 was used as the cut-off for progression-free survival. Mantel-Cox analysis was performed at Day 45.
To measure induction of T cell proliferation by DuoBody-PD-L1×4-1BB or Pembrolizumab, an antigen-specific T cell proliferation assay with active PD1/PD-L1 axis was performed.
HLA-A2+ peripheral blood mononuclear cells (PBMCs) were obtained from healthy donors (Transfusionszentrale, University Hospital, Mainz, Germany). Monocytes were isolated from PBMCs by magnetic-activated cell sorting (MACS) technology using anti-CD14 MicroBeads (Miltenyi; cat. no. 130-050-201), according to the manufacturer's instructions. The peripheral blood lymphocytes (PBLs, CD14-negative fraction) were frozen for future T-cell isolation. For differentiation into immature DCs (iDCs), 1×106 monocytes/mL were cultured for five days in RPMI GlutaMAX (Life technologies GmbH, cat. no. 61870-044) containing 5% human AB serum (Sigma-Aldrich Chemie GmbH, cat. no. H4522-100ML), sodium pyruvate (Life technologies GmbH, cat. no. 11360-039), non-essential amino acids (Life technologies GmbH, cat. no. 11140-035), 100 IU/mL penicillin-streptomycin (Life technologies GmbH, cat. no.15140-122), 1000 IU/mL granulocyte-macrophage colony-stimulating factor (GM-CSF; Miltenyi, cat. no. 130-093-868) and 1000 IU/mL interleukin-4 (IL-4; Miltenyi, cat. no. 130-093-924). Once during these five days, half of the medium was replaced with fresh medium. iDCs were harvested by collecting non-adherent cells and adherent cells were detached by incubation with PBS containing 2 mM EDTA for 10 min at 37°. After washing iDCs were frozen in RPMI GlutaMAX containing 10% v/v DMSO (AppliChem GmbH, cat. no A3672,0050)+50% v/v human AB serum for future antigen-specific T cell assays.
One day prior to the start of an antigen-specific CD8+ T cell proliferation assay, frozen PBLs and iDCs, from the same donor, were thawed. CD8+ T cells were isolated from PBLs by MACS technology using anti-CD8 MicroBeads (Miltenyi, cat. no. 130-045-201), according to the manufacturer's instructions.
About 10-15×106 CD8+ T cells were electroporated with 10 μg of in vitro translated (IVT)-RNA encoding the alpha-chain plus 10 μg of IVT-RNA encoding the beta-chain of a claudin-6-specific murine TCR (HLA-A2-restricted; described in WO 2015150327 A1) plus 10 μg IVT-RNA encoding PD-1 in 250 μL X-Vivo15 (Biozym Scientific GmbH, cat. no.881026) in a 4-mm electroporation cuvette (VWR International GmbH, cat. no. 732-0023) using the BTX ECM® 830 Electroporation System device (BTX; 500 V, 1×3 ms pulse). Immediately after electroporation, cells were transferred into fresh IMDM medium (Life Technologies GmbH, cat. no. 12440-061) supplemented with 5% human AB serum and rested at 37° C., 5% CO2 for at least 1 hour. T cells were labeled using 1.6 μM carboxyfluorescein succinimidyl ester (CFSE; Invitrogen, cat. no. C34564) in PBS according to the manufacturer's instructions, and incubated in IMDM medium supplemented with 5% human AB serum, O/N.
Up to 5×106 thawed iDCs were electroporated with either 1 μg (GEN1046 dose-response) or 3 μg (Pembrolizumab dose-response) IVT-RNA encoding full length claudin-6, in 250 μL X-Vivo15 medium, using the electroporation system as described above (300 V, 1×12 ms pulse) and incubated in IMDM medium supplemented with 5% human AB serum, O/N.
The next day, cells were harvested. Cell surface expression of claudin-6 and PD-L1 on DCs and TCR and PD-1 on T cells was checked by flow cytometry. DCs were stained with an Alexa647-conjugated CLDN6-specific antibody (non-commercially available; in-house production) and with anti-human CD274 antibody (PD-L1, eBioscienes, cat. no.12-5983) and T cells were stained with an anti-Mouse TCR B Chain antibody (Becton Dickinson GmbH, cat. no. 553174) and with anti-human CD279 antibody (PD-1, eBioscience, cat. no. 17-2799). Electroporated DCs were incubated with electroporated, CFSE-labeled T cells in a ratio of 1:10 in the presence of GEN1046 (at 3-fold serial dilutions from 1 to 0.00015 μg/mL) or clinical-grade Pembrolizumab (at 4-fold serial dilutions from 0.8 to 0.00005 μg/mL; Keytruda, Phoenix Apotheke, PZN 10749897) in IMDM GlutaMAX supplemented with 5% human AB serum in a 96-well round-bottom plate. Flow cytometric analysis of T cell proliferation based on CFSE-dilution was performed after 5 days on a BD FACSCanto™ II or BD FACSCelesta™ flow cytometer (Becton Dickinson GmbH). Acquired data was analyzed using FlowJo software version 10.7.1. The expansion index values (determines the fold-expansion of the overall culture) per treatment condition were calculated and plotted as a function of the GEN1046 or Pembrolizumab concentration. Dose-response curves were generated and EC20, EC50, EC90 and Hill-Slope values were calculated in GraphPad Prism version 9 (GraphPad Software, Inc.) using a 4-parameter logarithmic fit.
The GEN1046 dose response was analyzed at 3-fold serial dilutions from 1 to 0.00015 μg/mL (
The Pembrolizumab dose response was analyzed at 4-fold serial dilutions from 0.8 to 0.00005 μg/mL (
To measure induction of T cell proliferation by GEN1046 in combination with anti-PD-1 antibody Pembrolizumab or IgG1-ctrl antibody, an antigen-specific T cell proliferation assay with active PD1/PD-L1 axis was performed (general assay set-up analogous to example 1). In short, claudin-6-IVT-RNA electroporated DCs were incubated with claudin-6-specific TCR- and PD1-IVT-RNA electroporated, CFSE-labeled T cells (ratio of 1:10) in the presence of GEN1046 in combination with a fixed concentration of Pembrolizumab or isotype control antibody IgG1-ctrl in IMDM GlutaMAX supplemented with 5% human AB serum in a 96-well round-bottom plate. Three different concentrations of GEN1046 were tested, representing optimal, half-maximal and sub-optimal effective concentrations determined in previous experiments (0.2 μg/mL>EC90; 0.0067 μg/mL≈EC50; 0.0022 μg/mL≈EC20, see Example 1, Table 1). Pembrolizumab and the IgG1-ctrl antibody were tested at a concentration of 0.8 μg/mL, a concentration well above the EC90 value for Pembrolizumab (see Example 1, Table 2). Medium and 0.8 μg/mL IgG1-ctrl only were used to determine baseline proliferation. Pembrolizumab (0.8 μg/mL) was used as additional checkpoint inhibition control. Flow cytometric analysis of T cell proliferation based on CFSE-dilution was performed after 5 days on a BD FACSCanto™ II or BD FACSCelesta™ flow cytometer (Becton Dickinson GmbH). Acquired data was analyzed using FlowJo software version 10.7.1. The expansion index values per treatment condition were calculated and plotted using GraphPad Prism version 9 (GraphPad Software, Inc.).
Incubation of PD-1 and claudin-6-specific TCR expressing CD8+ T cells with DCs expressing PD-L1 and cognate antigen resulted in a minimal proliferation induction with expansion index values slightly above 1 in the medium only and IgG1-ctrl treated cultures for all three donors tested (see
The study is an open-label, multi-center, phase 1/2a safety trial of GEN1046 (DuoBody® PD L1×4 1BB). The trial consists of 2 parts; a first-in-human (FIH) dose escalation (phase 1) and an expansion (phase 2a). The dose escalation evaluated GEN1046 in subjects with solid malignant tumors to determine the maximum tolerated dose (MTD) or maximum administered dose and/or the recommended phase 2 dose (RP2D).
The expansion further evaluates the safety, tolerability, PK, and anti-tumor activity of the selected dose(s) in select solid tumors expansion cohorts for non-small cell lung cancer (NSCLC) (PD-1/L1 pre-treated and PD-1/L1 naïve), urothelial cancer (UC), endometrial cancer (EC), triple negative breast cancer (TNBC) (in subjects who have received prior treatment with a PD-1/L1 inhibitor and in subjects who have not received such treatment); and squamous cell carcinoma of the head and neck (SCCHN).
A diagram of the trial design is provided in
Expansion Cohorts (EC) A and B: NSCLC Treatment-Naive for Metastatic Disease: GEN1046 in Combination with Pembrolizumab)
Expansion Cohorts ECA and ECB evaluates 100 mg GEN1046 in combination with pembrolizumab at 2 different dosing schedules: ECA tests a GEN1046 regimen of 100 mg 1Q3W with a pembrolizumab regimen of 200 mg 1Q3W.
Based on PK/pharmacodynamic modeling, this regimen of GEN1046 is expected to result in peak trimer formation and sustained 4-1BB activation, which in combination with pembrolizumab may allow for optimum engagement of both targets/pathways and improved anti-tumor efficacy.
ECB evaluates a GEN1046 regimen of 100 mg 1Q6W with apembrolizumab regimen of 400 mg 1Q6W. Based on PK/pharmacodynamic modeling, this regimen of GEN1046 is expected to provide intermittent/transient activation of 4-1BB in a 6-week dosing cycle compared to sustained 4-1BB activation in a 3-week dosing cycle. Transient activation of 4-1BB is expected to allow for resetting of the T-cell response and reduce chronic interferon signaling (Weber, E. W., et al. (2021), Science 372 (6537)), which may prevent exhaustion of tumor infiltrating CD8+ T cells due to continuous 4-1BB activation and, in combination with pembrolizumab, may provide improved depth and duration of response (DoR).
Pembrolizumab regimens of 200 mg Q3W and 400 mg Q6W have been approved as first- and second-line SOC treatment for NSCLC, respectively.
Treatment continues until the subject fulfills one of the treatment discontinuation criteria (please see below).
Expansion cohorts A and B
Any potential subject who meets any of the following criteria will be excluded from participating in the trial.
GEN1046 is administered using intravenous (IV) infusion over a minimum of 60 minutes on Day 1 of each treatment cycle of either 21 days or 42 days after all procedures and assessments have been completed.
In ECA and —B, subjects receive GEN1046 100 mg 1Q3W in combination with pembrolizumab 200 mg 1Q3W (ECA) or GEN1046 100 mg 1Q6W in combination with pembrolizumab 400 mg 1Q6W (ECB).
ECA and ECB: Pembrolizumab 200 mg or 400 mg is administered on Day 1 of each 3-week OR 6-week treatment cycle, respectively, after all pre-treatment procedures and assessments have been completed.
Pembrolizumab is administered first, followed by GEN1046. Pembrolizumab is administered as an intravenous infusion over 30 minutes. Pembrolizumab must be promptly followed by a saline flush to clear the line before starting the infusion of GEN1046. The time in between infusions is approximately 30 minutes or longer depending on the situation. Dose reductions for pembrolizumab are not recommended.
GEN1046—20 mg/mL formulated in 20 mM histidine, 250 mM Sucrose, 0.02% Polysorbate-80, pH 5.5—is a clear to opalescent, colorless to slightly yellow solution supplied as a concentrate for solution for infusion to be diluted (at site) in 0.9% NaCl (saline).
Pembrolizumab (Keytruda®) infusion is a sterile, preservative-free, clear to slightly opalescent, colorless to slightly yellow solution that requires dilution for IV infusion.
For subjects who experience an IRR associated with administration of GEN1046:
Pembrolizumab may cause severe or life-threatening IRRs including severe hypersensitivity or anaphylaxis. Signs and symptoms usually develop during or shortly after drug infusion and generally resolve completely within 24 hours of completion of infusion.
Subjects receive GEN1046 treatment on Day 1 of each 3-week or 6-week treatment cycle until one of the predefined discontinuation of treatment criteria (below) has been met.
Subjects in cohorts ECA and ECB receive pembrolizumab treatment in combination with GEN1046 on Day 1 of each 3-week treatment cycle or on Day 1 of each 6-week treatment cycle, respectively, until progressive disease, or until 1 of the predefined discontinuation of treatment criteria has been met. Both GEN1046 and pembrolizumab should be discontinued. Subjects may only continue on GEN1046 or pembrolizumab monotherapy if approved by sponsor's medical monitor. Subjects move into the safety follow-up period once both drugs have been discontinued.
All subjects have imaging of the brain, thorax, abdomen, and pelvis performed during screening. Head and neck imaging is always required for subjects with SCCHN.
Tumor imaging is preferably acquired by computed tomography (CT). Up to 5 target lesions (maximum 2 per organ) are defined at screening and are followed throughout the trial. Non-target lesions are also assessed throughout the trial. Initial tumor imaging at screening is performed within 21 days prior to the date of first dose. The site reviews screening images to confirm the subject has measurable disease per RECIST 1.1.
On-trial imaging is performed every 6 weeks (±7 days) for 50 weeks, and every 12 weeks (±7 days) thereafter from the date of first dose until disease progression is assessed by the investigator (unless the investigator elects to continue treatment and follow iRECIST), the start of new anti-cancer therapy, withdrawal of consent, or death, whichever occurs first.
RECIST 1.1 criteria are used for secondary endpoint response evaluation (Eisenhauer et al., 2009, Eur J Cancer 45, 228-247.); iRECIST are used for exploratory endpoint response evaluation (Seymour et al., 2017, Lancet Oncol 18, e143-e152). If the investigator elects to apply iRECIST, treatment should continue until PD has been verified.
Additional CT scans or MRI scans may be performed at the investigators discretion to confirm response or new symptoms. Tumor imaging to confirm PR or CR should be performed at least 4 weeks after the first indication of a response is observed.
iRECIST Assessment of Disease
iRECIST is based on RECIST 1.1 but has been modified to account for the unique response patterns observed with immunotherapy. In this trial, iRECIST is evaluated as an exploratory endpoint (Seymour et al., 2017, Lancet Oncol 18, e143-e152).
iRECIST disease progression should be confirmed at least 4 to 7 weeks after the first radiologic evidence of PD in clinically stable participants. Subjects who have unconfirmed disease progression may continue on GEN1046 treatment until progression is confirmed as long as the subject is clinically stable. Subjects who are clinically stable must meet the following criteria:
Any clinically unstable subjects are discontinued from GEN1046 treatment at the first occurrence of radiographic disease progression. If repeat imaging shows iRECIST confirmed disease progression (iCPD), subjects will discontinue GEN1046 treatment.
The ECOG performance status will be assessed by the investigator at screening, on Day 1 of each cycle, and at the treatment discontinuation visit. Performance status will be scored using the ECOG performance status scale index (Table 12).
The trial is a phase 2, multicenter, randomized, open-label trial evaluating the safety and efficacy of GEN1046 as monotherapy and in combination therapy with pembrolizumab in adult subjects with locally advanced or metastatic NSCLC after treatment with CPI-containing therapy.
Approximately 126 subjects will be enrolled in the trial, and 120 eligible subjects (40 in each arm) will be randomized to one of the treatment arms described below. Subjects must provide fresh and/or archival tumor tissue for prospective central confirmation of PD-L1 expression in the tumors. The percentage of subjects with non-squamous histology will be capped at approximately 70%. Randomization will be stratified by PD-L1 expression (TPS ≥50% vs 1% to 49%) and histology (squamous vs non-squamous).
During a preliminary safety run-in, 6 subjects will be enrolled to Arms B and C (3 subjects per arm). These subjects will be closely monitored and followed for a minimum of 3 weeks. After completion of the safety run-in for these cohorts, the collected data (including, but not limited to, all relevant safety and clinical data) will be evaluated. After this review, if the combination regimen is considered well tolerated, randomization for Arms A, B, and C will begin.
Treatment for a subject should continue until the subject fulfils one of the treatment discontinuation criteria defined below.
Computed tomography (CT) with contrast or magnetic resonance imaging (MRI) is obtained at baseline before the first dose and 6, 12, 18, and 24 weeks (±7 days) after the first dose of the trial medication, and thereafter, every 9 weeks (±7 days). CT or MRI will continue to be obtained until disease progression (as assessed by the investigator), start of subsequent anticancer therapy, withdrawal of consent, or death, whichever occurs first. Response Evaluation Criteria in Solid Tumors (RECIST) v1.1-defined disease progression must be confirmed by an additional confirmatory scan following the initial documented progressive disease (PD). Any clinically unstable subject is discontinued from trial treatment at the first occurrence of radiographic disease progression and is not required to have repeated imaging to confirm PD. Should delayed response to treatment be suspected, the investigator may continue treatment beyond the time of RECIST v1.1-defined progression, if the subject is experiencing clinical benefit. Once a subject experiences PD, survival status is collected every 12 weeks until death, withdrawal of consent, loss to follow up, or the end of the trial, whichever occurs first. Subsequent anti-cancer treatments and the subject's response to them are also collected. During treatment beyond RECIST v1.1-defined progression, iRECIST is used to assess subsequent progression.
The trial is a randomized, open-label trial evaluating the safety and efficacy of GEN1046 as monotherapy or in combination with pembrolizumab in adult subjects with relapsed/refractory metastatic NSCLC after treatment with CPI-containing therapy. Randomization is used to eliminate potential allocation bias while an open-label design will allow efficient AE/SAE management. PD L1 expression level is associated with efficacy for pembrolizumab and histology is an important baseline disease characteristic. Randomization will be stratified to ensure balance across arms for these 2 factors.
The primary objective is to evaluate the anti-tumor activity objective response rate (ORR) of GEN1046 as monotherapy and in combination with pembrolizumab. ORR is a well-established efficacy parameter for assessing anti-tumor activity in a proof-of-concept trial in NSCLC.
The selection of the 100 mg Q3W dose for GEN1046 was based on clinical data from the FIH trial, GCT1046-01, where doses ranging from 25 to 1200 mg Q3W were evaluated in 61 subjects in the dose-escalation phase. In addition, a PK/pharmacodynamic model was developed to predict 4-1BB, GEN1046, PD-L1 trimolecular complex (trimer) formation, and RO for PD-L1 in tumors to understand the PK/pharmacodynamic/efficacy relationship (see Example 9).
In summary, the GEN1046 doses of 100 mg Q3W was selected based on the dose escalation study in Example 7.
Arm A will test a regimen of an activation dose of GEN1046 (100 mg Q3W for 2 cycles) followed by a higher maintenance dose of GEN1046 (500 mg administered Q6W for the subsequent cycles), based on the following:
Arms B and C will evaluate GEN1046 in combination with pembrolizumab at 2 different dosing schedules:
Pembrolizumab regimens of 200 mg Q3W and 400 mg Q6W have been approved as first- and second-line SOC treatment for NSCLC, respectively. Pembrolizumab 200 mg Q3W and 400 mg Q6W are expected to provide comparable efficacy and safety profiles (Lala et al., 2020, Eur J Cancer 131, 68-75).
Any potential subject who meets any of the following criteria will be excluded from participating in the trial.
Trial treatments are administered as described in Table 13, until 1 or more of the discontinuation criteria below are met.
GEN1046 and pembrolizumab will be administered as IV infusions by qualified site personnel. During drug product preparation and handling, vigorous mixing or shaking is to be avoided. Care must be taken to assure sterility of the prepared solution as the product does not contain any antimicrobial preservative or bacteriostatic agent.
In Arm A, GEN1046 100 mg Q3W is administered as a 30-minute IV infusion on Day 1 for the first 2 treatment cycles; thereafter, GEN1046 500 mg Q6W is administered as a 30-minute IV infusion on Day 1 of the subsequent 6-week treatment cycles. No dose reduction is allowed for GEN1046.
In Arm B, GEN1046 100 mg and pembrolizumab 200 mg will be administered on Day 1 of every 3-week cycle.
In Arm C, GEN1046 100 mg and pembrolizumab 400 mg will be administered on Day 1 of every 6-week cycle.
Pembrolizumab is administered first followed by GEN1046. Pembrolizumab is promptly followed by a saline flush to clear the line before starting the infusion of GEN1046. The time in between infusions is approximately 30 minutes or longer depending on the situation. Dose reductions for GEN1046 and/or pembrolizumab are not allowed.
GEN1046—20 mg/mL formulated in 20 mM histidine, 250 mM Sucrose, 0.02% Polysorbate-80, pH 5.5—is a clear to opalescent, colorless to slightly yellow solution supplied as a concentrate for solution for infusion to be diluted (at site) in 0.9% NaCl (saline).
Pembrolizumab (Keytruda®) infusion is a sterile, preservative-free, clear to slightly opalescent, colorless to slightly yellow solution that requires dilution for IV infusion.
A subject's trial treatment must be discontinued in the event of:
Survival status is assessed every 12 weeks (±14 days), beginning from the day of last GEN1046 dose and continuing until the subject dies or withdraws from the trial. Subjects who are not available, or whose designated family members are not available, for this assessment are entered as “lost to follow up”
Subject Withdrawal from the Trial
A subject will be withdrawn from the trial for any of the following reasons:
If a subject discontinues trial treatment and withdraws from the trial before demonstrating PD, the end-of-treatment assessments should be obtained.
Tumor response is assessed locally according to the RECIST v1.1 criteria (Eisenhauer et al., 2009). The imaging assessment collection plan is presented below. In addition, prior tumor scan images are collected if feasible.
Imaging data are centrally collected and checked for quality by an imaging contract research organization (CRO) designated by the sponsor. The local investigator's assessment will be used for the primary endpoint analysis and for treatment decision-making.
For subjects who continue trial treatment beyond initial RECIST-defined disease progression, efficacy assessment must continue.
For subjects who discontinue trial treatment without RECIST-defined disease progression as assessed by the investigator, on-trial imaging must continue until RECIST-defined disease progression, start of subsequent anti-cancer therapy, withdrawal of consent, death, or loss to follow up, whichever occurs first.
Imaging assessments are performed at screening/baseline within 21 days of administration of the first dose of trial treatment (Day −21 to Day −1 prior to CID1). All sites of metastatic disease are reported as target or non-target lesions at baseline and followed throughout the trial.
Any imaging assessments already completed during the regular evaluation of the subject within 21 days prior to start of treatment, including before signing the main trial ICF, can be considered as the baseline images for this trial only if they fulfill the technical imaging requirements for the trial. Any imaging assessments obtained after randomization cannot be considered baseline images.
All scheduled tumor imaging, including screening, must include complete imaging of the chest, abdomen, and pelvis. Tumor imaging is strongly preferred to be acquired by CT with iodinated contrast. For the abdomen and pelvis, MRI may be used when CT with iodinated contrast is contraindicated, or when local practice mandates it. Chest imaging must be done by CT, but may be done without contrast when iodinated contrast is contraindicated.
MRI is the strongly preferred modality for imaging the brain. In case of known or suspected brain metastases, brain MRI is completed at baseline. Contrast-enhanced brain MRI is preferred; however, if MRI contrast is contraindicated, then MRI without contrast or CT with/without contrast is acceptable.
Any potentially measurable lesion that has been previously treated with radiotherapy should be considered as a non-measurable lesion. However, if a lesion previously treated with radiotherapy has clearly progressed since the radiotherapy, it can be considered as a measurable lesion.
Imaging assessments as described in Table 14 are performed using the same imaging modality used at baseline, irrespective of trial treatment interruption or actual dosing. On-trial imaging is performed every 6 weeks (±7 days) for the first 24 weeks, and every 9 weeks (±7 days) thereafter from the date of first dose until disease progression (as assessed by the investigator), start of subsequent anti-cancer therapy, withdrawal of consent, death, or loss to follow up, whichever occurs first. Imaging assessments are scheduled using the date of first dose of trial treatment as the reference, and are respected regardless of whether treatment with trial drug is temporarily withheld or unscheduled assessments performed.
Additional imaging assessments may be performed at any time during the trial at the investigator's discretion to support the efficacy evaluations for a subject, as necessary. Clinical suspicion of disease progression at any time requires a physical examination and imaging assessments to be performed promptly rather than waiting for the next scheduled imaging assessment.
Each lesion that is assessed at baseline must be assessed by the imaging method and when possible, the same local radiologist/physician throughout the trial so that the comparison is consistent. If an off-schedule imaging assessment is performed because progression is suspected, subsequent imaging assessments should be performed in accordance with the original imaging schedule.
Combined positron emission tomography (PET)-CT may be used only if the CT is of similar diagnostic quality as a CT performed without PET, including the utilization of IV contrast media. At the discretion of the investigators, FDG-PET scans may be performed to document PD as per RECIST 1.1.
Progression-free survival 2 (PFS2) is defined as time from first infusion to objective tumor progression on next-line treatment or death from any cause. For PFS2, objective tumor progression will be determined based on investigator assessment of progression on next-line therapy. For this purpose, subsequent anti-neoplastic therapies including start/end date, reason for discontinuation, and date of disease progression will be captured.
iRECIST Assessment of Disease
iRECIST disease progression should be confirmed at least 4-7 weeks after the first radiologic evidence of PD in clinically stable participants. Subjects who have unconfirmed disease progression may continue on trial treatment until progression is confirmed as long as the subject is clinically stable.
Subjects who are clinically stable must meet the following criteria:
Any clinically unstable subjects are discontinued from trial treatment at the first occurrence of radiographic disease progression. Subjects that are clinically unstable are not required to have repeated imaging to confirm PD by iRECIST; however, a confirmation of progression scan may be obtained at the investigator's discretion after consultation with the sponsor.
If repeat imaging shows iRECIST-confirmed disease progression (iCPD), subjects will discontinue trial treatment. However, if the subject is deriving benefit after iCPD is observed, an exception to continue trial treatment must be approved by the sponsor medical monitor. If repeat imaging shows iRECIST stable disease (iSD), iRECIST partial response (iPR), or iRECIST complete response (iCR), imaging should be continued every 6 weeks (±7 days) and the subject should continue on trial treatment.
As of 10 Aug. 2022, 4 patients have been enrolled in the safety run-in part of the Arm C and DLT period of the 4th patient has been completed. No DLTs has been reported in the evaluable subjects, and none of the reported SAEs were related to study drug (GEN1046 or Pembrolizumab). 1 subject experienced partial response and 1 subject has stable disease.
An integrated quantitative systems pharmacology (QSP) model was developed that combines a multi-compartment physiologically-based pharmacokinetic (PBPK) model with a mechanistic systems biology model representing the tumor/immune processes and interactions that occur in the tumor micro-environment and draining lymph nodes. The PBPK model describes FcRn-mediated GNE1046 transport and distribution to healthy tissue spaces and tumor spaces, with return to plasma mediated by lymphatic flow. The mechanistic systems biology model is designed to recapitulate the known biology involved in tumor proliferation, the cancer/immunity cycle, and the mechanisms directly related to the activity of GEN1046 and an anti-PD1 drug Pembrolizumab. The model leveraged a wide range of literature, preclinical, pharmacokinetic, and pharmacodynamic data for parameterization of these pathways. The model was used to generate a population of virtual patients spanning observed variability in key markers (e.g., PD-1 and PD-L1 expression at baseline) and was used to predict objective response rate (ORR) when GEN1046 and Pembrolizumab given as monotherapy or in combination. Model was validated based on objective response rate (ORR) observed for Pembrolizumab (data from literature) and GEN1046 monotherapy clinical trials. Evaluation was done for GEN1046 at 100 mg Q3W or 100 mg Q6W given as monotherapy or in combination with Pembrolizumab at an approved dose of 200 mg Q3W or 400 mg Q6W.
A weak dose-dependent relationship was observed in both partial response (PR) and complete (CR) rates for GEN1046 given as 100 mg Q3W or Q6W in combination with Pembrolizumab. Prediction for PR and CR rates shows maximum response between GEN1046 100 mg and 300 mg Q3W. Additionally, predictions show minimal differences between GEN1046 and Pembrolizumab when given Q3W or Q6W (
Objective: To analyze if the combination of GEN1046 with pembrolizumab could reverse T-cell exhaustion in a mixed lymphocyte reaction (MLR) assay, four unique, allogeneic pairs of human mature dendritic cells (mDCs) and in vitro exhausted T cells (Tex) were co-cultured in the presence of GEN1046 alone, pembrolizumab alone, or a combination of both antibodies. Expression of inhibitory receptors on Tex was determined by flow cytometry and secretion of interferon (IFN)γ was assessed in the supernatants of the co-cultures.
Monocytes and T Cells from Healthy Donors
CD14+ monocytes and purified CD3+ T cells were obtained from BioIVT. Four unique allogeneic donor pairs were used for the MLR assay.
Human CD14+ monocytes were obtained from healthy donors. For differentiation into immature dendritic cells (iDCs), 1-1.5×106 monocytes/mL were cultured for six days in Roswell Park Memorial Institute (RPMI) 1640 complete medium (ATCC modification formula; ThermoFisher, cat. no. A1049101) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco, cat. no. 16140071), 100 ng/mL granulocyte-macrophage colony-stimulating factor (GM-CSF; BioLegend, cat. no. 766106) and 300 ng/mL interleukin (IL)-4 (BioLegend, cat. no. 766206) in T25 culture flasks (Falcon, cat. no. 353108) at 37° C. After four days, the medium was replaced with fresh medium and supplements.
Differentiation of iDCs to mDCs
Prior to start of the MLR assay, iDCs were harvested by collecting non-adherent cells and differentiated to mDCs by incubating 1-1.5×106 cells/mL in RPMI 1640 complete medium supplemented with 10% FBS, 100 ng/mL GM-CSF, 300 ng/mL IL-4 and 5 μg/mL lipopolysaccharide (LPS; ThermoFisher, cat. no. 00-4976-93) for 24 h at 37° C.
Purified CD3+ T cells obtained from healthy donors were thawed and resuspended at 1×106 cells/mL in AIM-V medium (ThermoFisher, cat. no. 12055091) supplemented with 5% FBS and 10 ng/mL IL-2 (BioLegend, cat. no. 589106). To generate T cells with an exhausted-like phenotype, the cells were stimulated for two rounds with of Dynabeads™ Human T Activator CD3/CD28 (Gibco, cat. No. 11161D) at a bead:cell ratio of 1:1 for 48 h at 37° C. and 5% CO2. The exhausted phenotype of the T cells was confirmed by hyporesponsiveness to CD3/CD28 restimulation (lack of IFNγ secretion), as described below. High expression of the inhibitory receptors TIM3, LAG3 and PD-1 was consistent with an exhausted phenotype. After two rounds of stimulation, the exhausted CD3+ T cells (Tex) were rested for 24 h.
As a naïve control, purified CD3+ T cells obtained from healthy donors were thawed one day prior to the start of the MLR assay, resuspended at 1×106 cells/mL in RPMI 1640 complete medium supplemented with 10% FBS and 10 ng/mL IL-2 and incubated O/N at 37° C. Prior to the MLR assay, aliquots of naïve T cells and Tex were collected for flow cytometry.
For flow cytometry analysis of inhibitory receptors on Tex, cells were pelleted at 400×g for 5 min, washed in phosphate-buffered saline (PBS), pelleted again, resuspended in 1 mL PBS supplemented with LIVE/DEAD™ Fixable Near-IR Dead Cell Stain (ThermoFisher Scientific, cat. no. L10119, diluted 1:500) or Viability Live/Dead Blue (ThermoFisher Scientific, cat. no. L2305, diluted 1:500) and incubated for 20 min at 4° C. in the dark. Next, cells were washed, pelleted, resuspended to 8×106 cells/mL in FACS buffer (Dulbecco's phosphate-buffered saline [DPBS, Gibco, cat. no. 14190136] supplemented with 0.5% bovine serum albumin [BSA, Sigma, cat. no. A9576] and 2 mM ethylenediaminetetraacetic acid [EDTA, Invitrogen, cat. no. 15575-038]) containing 5% human serum (Sigma, cat. no. H4522), and incubated for 15 min at 4° C. Then 25 μL containing 2×105 cells was transferred to a new 96-well plate containing 150 μL staining mix with fluorescently-labeled antibodies shown in Table 15 diluted in FACS buffer supplemented with Brilliant Stain Buffer Plus (BD Horizon, cat. no. 566385) and incubated for 20 min at RT in the dark. Cells were pelleted, washed using FACS buffer, resuspended in 100 μL Fixation Buffer (Biolegend, cat. no. 420801) and incubated for 15 min at 4° C. in the dark. Cells were pelleted again, washed and resuspended in 100 μL FACS buffer. Samples were analyzed on a Cytek® Aurora flow cytometer (Cytek Biosciences).
The mDCs (see Differentiation of iDCs to mDCs) were harvested and resuspended in AIM-V medium at 4×105 cells/mL. Tex and naïve CD3+ T cells (see Exhaustion of T cells) were harvested and resuspended in AIM-V medium at 4×106 cells/mL. Co-cultures of mDC and Tex were seeded at a DC:T cell ratio of 1:4 or 1:10, corresponding to 2×104 mDCs incubated with 8×104 or 2×105 Tex, and cultured in the presence of pembrolizumab (1 μg/mL; non-clinical/research-grade version of the clinical product pembrolizumab; Selleckchem, cat. no. A2005) or GEN1046 (0.001-30 μg/mL) as single agent, or both agents combined in AIM-V medium in a 96-well round-bottom plate (Falcon, cat. no. 353227) at 37° C. for 5 days. Co-cultures treated with bsIgG1-PD-L1×ctrl (30 μg/mL), bsIgG1-ctrl×4-1BB (30 μg/mL), IgG1-ctrl-FEAL (30 μg/mL) or IgG4 isotype control (1 μg/mL) were included as controls (Table 16). In parallel, co-cultures of mDC and naïve CD3+ T cells at a DC:T cell ratio of 1:10, corresponding to 2×104 mDCs incubated with 2×105 T cells, were cultured with and without 1 μg/mL pembrolizumab. After 5 days, the plates were centrifuged at 500×g for 5 min and the supernatant was carefully transferred from each well to a new 96-well round bottom plate.
The collected supernatants were analyzed for IFNγ levels by enzyme-linked immunosorbent assay (ELISA) using an AlphaLISA IFNγ kit (Perkin Elmer, cat. no. AL217) on an Envision instrument, according to the manufacturer's instructions.
1Control binding moiety based on anti-HIV gp120 antibody IgG1-b12 (Barbas et al., J Mol Biol 1993, 230: 812-823)
The cytokine concentration values in each treatment condition were normalized by subtracting the background control values (no treatment control wells) and expressed as a percentage of the maximal value in the assay. The combination effect was quantified by comparing the observed response against the expected response using the Highest Single Agent (HSA) reference model, which is defined as the maximum single drug response at corresponding concentrations.
After two rounds of stimulation with CD3/CD28 beads, the T cells became hyporesponsive to dual anti-CD3 and anti-CD28 stimulation, consistent with an exhausted phenotype as demonstrated by reduced secretion of IFNγ (
Objective: To investigate the anti-tumor activity of mbsIgG2a-PD-L1×4-1BB antibody either alone or in combination with anti-mPD-1 in the MC38 colon cancer model in C57BL/6 mice.
MC38 mouse colon cancer cells were cultured in Dulbecco's Modified Eagle Medium supplemented with 10% heat-inactivated fetal bovine serum at 37° C., 5% CO2. MC38 cells were harvested from a cell culture growing in log-phase and quantified.
MC38 cells (1×106 tumor cells in 100 μL PBS) were injected subcutaneously in the right lower flank of female C57BL/6 mice (obtained from Shanghai Lingchang Biotechnology Co., Ltd and Services; age 6-8 weeks at start of experiment).
Tumor growth was evaluated three times per week using a caliper. Tumor volumes (mm3) were calculated from caliper measurements as ([length]×[width]2)/2, where the length is the longest tumor dimension and the width is the longest tumor dimension perpendicular to the length.
Treatment was initiated when tumors had reached a mean volume of 60 mm3. Mice were randomized into groups (n=10/group) with equal mean tumor volume prior to treatment. On treatment days (two doses weekly for three weeks [2QW×3]), the mice were injected intraperitoneally with the antibodies indicated in Table 17 in an injection volume of 10 μL/g body weight. For combination treatments, antibodies were injected in two separate injections with 20 min in between (Table 17). Dose levels were based on previous experience with these antibodies in the MC38 mouse model.
The mice were monitored daily for clinical signs of illness. Body weight measurements were performed three times a week after randomization. The antibodies and combinations thereof were well tolerated, as mice showed minimal body weight loss (<20%) upon treatment, rather an increase in body weight. The experiment ended for the individual mice when the tumor volume exceeded 1500 mm3 or when the animals reached humane endpoints (e.g. when mice showed body weight loss >20%, when tumors showed ulceration [>75%], when serious clinical signs were observed and/or when the tumor growth blocked the physical activity of the mouse).
ambsIgG2a-PD-L1 × 4-1BB was injected first and the second antibody was injected after 20 min
Mice that showed complete regression of tumors after antibody treatment were rechallenged with MC38 tumor cells 121 days after treatment initiation. Mice were inoculated with 1×106 fresh MC38 tumor cells on the opposite flank of the original tumor cell inoculation. As control treatment of tumor outgrowth, a group of age matched naïve C57BL/6 mice (n=6) was inoculated with MC38 tumor cells from the same cell culture.
Rapid tumor outgrowth was observed in MC38-bearing mice treated with nonbinding control antibody mIgG2a-ctrl-AAKR (5 mg/kg;
Mice with complete tumor regression, eg, where the tumors disappeared completely for the duration of the observation period (Table 18), and a control group of six age-matched tumor-naïve mice, were (re)challenged with MC38 tumor cells that were SC injected on Day 121 after the treatment with antibodies was initiated. A control group of six age-matched tumor-naïve mice was SC injected with MC38 tumor cells at the same time. In all naïve mice, the MC38 tumor grew out to 1,500 mm3 at Day 24 after tumor inoculation, whereas there was no tumor outgrowth observed in the rechallenged mice during the entire follow-up period of 35 days after the rechallenge (156 days after the original inoculation with MC38 tumor cells), consistent with the development of immune memory (
These results provide rationale for evaluating the combination of GEN1046 with an anti-PD-1 antibody to further amplify the anti-tumor immune response in cancer patients to produce durable and deep clinical responses and enhance survival.
1Tumor volume < 500 mm3 was used as the cut-off for progression-free survival. Mantel-Cox analysis was performed at Day 69.
2A p-value < 0.05 was considered significant
Objective: As described in Example 2 and 11, mbsIgG2a-PD-L1×4-1BB combined with anti-mPD-1 showed potent anti-tumor activity with a durable response in the MC38 colon cancer model in C57BL/6 mice. Therefore, this model was used to further study the mechanism of action of the combination of mbsIgG2a-PD-L1×4-1BB and anti-mPD-1 in vivo. MC38-bearing mice were treated with mbsIgG2a-PD-L1×4-1BB, anti-mPD-1 or the combination thereof.
MC38 Colon Cancer model
MC38 mouse colon carcinoma tumors from two independent studies were collected for immunohistochemistry and flow cytometry assessments to characterize the in vivo activity of mbsIgG2a-PD-L1×4-1BB and anti-mPD-1 as monotherapy and in combination.
The MC38 tumor model was established as described in Examples 2 and 11. Treatment of mice bearing MC38 SC tumors was initiated when tumors had reached a tumor volume of 50-70 mm3. Mice were randomized into groups with equal mean tumor volume prior to treatment. On treatment days (two doses weekly for two weeks [2QW×2]), the mice were injected intraperitoneally with the antibodies indicated in Table 20 in an injection volume of 10 μL/g body weight. For combination treatments, antibodies were injected in two separate injections with 20 min in between (Table 20).
The mice were monitored daily for clinical signs of illness. Body weight measurements were performed three times a week after randomization. On Day 7 or 14 after initiation of treatment, mice (n=5 per group) were euthanized for resection of the tumors.
ambsIgG2a-PD-L1 × 4-1BB was injected first and the second antibody was injected after 20 min
Tumors were dissected, fixed in formalin, paraffin embedded and sectioned (4 m). For histologic assessment, tumor sections were deparaffinized and stained with the Tissue-Tek Prisma H&E Stain Kit (Sakura [Torrance, CA], 6190) using the Tissue-Tek Prisma Plus Automated Slide Stainer (Sakura). For evaluation of CD3+, CD4+ and CD8+ cells within the tumor, sections were deparaffinized and antigens were retrieved using CC1 buffer (Roche, 950-124), followed by quenching of endogenous peroxidase (Dako Agilent, S2003) and blocking of aspecific binding sites with blocking buffer (Roche, 05268869001) using the Roche Ventana Discovery (DISC) autostainer platform. Sections were incubated with primary antibodies (listed in Table 21), which were detected using anti-rabbit immunohistochemistry detection kits: for CD3 and CD4 with only anti-rabbit DISC, Omnimap (Roche, 05269679001) for CD8 sequentially with DISC anti-rabbit HQ (Roche, 07017812001) and DISC, and amplification for anti-HQ HRP Multimer (Roche, 06442544001). HRP was visualized using 3,3′-diaminobenzidine (ChromoMap DAB; Roche, 05266645001) according to manufacturer instructions. For evaluation of PD-L1+ cells within the tumor, sections were deparaffinized and antigens were retrieved using ER2 buffer (Leica Biosystems, AR9640), followed by quenching of endogenous peroxidase (Dako Agilent, S2003) and blocking aspecific binding sites with blocking buffer (Leica Biosystems, DS9800) using the Leica Bond Rx autostainer platform. Sections were incubated with the primary antibody (listed in Table 21), which were detected using anti-rabbit immunohistochemistry detection kit (Leica Biosystems, DS9800) according to manufacturer instructions. For evaluation of 4-1BB+ and PD-L2+ cells within the tumor, RNAscope assays have been performed on Leica Bond Rx with corresponding RNAscope probes (ACDBio, 493658 and 447788, respectively) and RNAscope detection kits (ACDBio, 322150) for detection of gene-specific mRNA molecules. In all assays, nuclei were counterstained by incubation with Mayer hematoxylin. Staining specificity was controlled by incorporating isotype, positive and negative control staining on consecutive tissue sections. Stained slides were subjected to whole slide imaging (Zeiss, Axioscan) and whole slide images were uploaded to and analyzed with Halo software (Indica Labs, Albuquerque, NM) using preprogrammed software analysis tools to determine CD3+, CD4+, CD8+ and PD-L1+ cells (CytoNuclear v2.0.9) and to determine 4-1BB+ and PD-L2+ cells (ISH v4.1.3). Quantitative data on CD3+, CD4+, CD8+, and PD-L1+ cells were subsequently expressed as percentage of marker-positive cells in relation to total cell numbers. Quantitative data on 4-1BB+ and PD-L2+ cells were expressed as RNAscope H-scores by creating four RNAscope intensity buckets and calculating H-scores with the formula: H-score=[(0×% cells with 0 dots/cell)+(1×% cells with 1-3 dots/cell)+(2×% cells with 4-9 dots/cell)+(3×% cells with 10-15 dots/cell)+(4×% cells with >15 dots/cell)].
Dissociated tumor cells were blocked with 1 μg/mL Mouse BD Fc Block™ (Fc blocking buffer; BD, cat. no. 553141) at 4° C. in the dark for 10 min. For staining of cell surface markers, the fluorescently-labeled antibody mixture described in Tables 22 (except Ki67 and GzmB) diluted in Fc blocking buffer were added to the cells, and incubated at 4° C. for 30 min, protected from light. For intracellular staining (Ki67 and GzmB), the cells were permeabilized by incubation with 200 μL Fix/Perm concentrate (eBioscience, cat. no. 00-5123) diluted in Fix/Penn dilution buffer (1:4; eBioscience, cat. no. 00-5223) at RT for 30 min, protected from light. After washing twice in Permeabilization buffer (eBioscience, cat. no. 00-8333), cells were incubated with Ki67 and GzmB antibodies (Tables 22) diluted in Permabilization buffer at RT for 30 min, protected from light. Finally, cells were resuspended in 250 μL FACS buffer (PBS supplemented with 10% FBS [Gibco, cat, no. 10099-141] and 40 mM EDTA [Boston BioProducts, cat no. BM-711-K]) and measured at the BD LSRFortessa™ X20 cell analyzer (BD Biosciences, San Jose, CA, USA). Data were analyzed using Kaluza Analysis Software.
Tumor tissue sections were evaluated for T cell subsets and target expression by immunohistochemistry (IHC) and in situ hybridization (ISH) on day 7 and day 14 following treatment initiation (
Treatment with mbsIgG2a-PD-L1×4-1BB and anti-mPD-1 as single agents enhanced the percentage of CD3+ cells within the tumor on Day 7 and Day 14 post-treatment. The combination of mbsIgG2a-PD-L1×4-1BB with anti-mPD-1 further increased the percentage of CD3+ cells on Day 14 (
The percentage of CD8+ cells was increased by mbsIgG2a-PD-L1×4-1BB compared to the PBS group on both Day 7 and Day 14, but not by anti-mPD-1. The combination mbsIgG2a-PD-L1×4-1BB with anti-mPD-1 showed similar levels of CD8+ cells compared to mbsIgG2a-PD-L1×4-1BB alone, suggesting that the increase in CD8+ cells was driven by mbsIgG2a-PD-L1×4-1BB (
On Day 7 and/or Day 14, intratumoral PD-L1 and PD-L2 expression was increased by mbsIgG2a-PD-L1×4-1BB and anti-mPD-1 as single agents compared to the PBS-treated mice. By contrast, the combination of mbsIgG2a-PD-L1×4-1BB with anti-mPD-1 did not show such an increase, as the levels of intratumoral PD-L1 and PD-L2 were comparable to the levels in PBS-treated mice (
In dissociated tumor tissues, it was found that the percentage of GzmB+ within the total intratumoral CD8+ T cell population was significantly enhanced by the combination of mbsIgG2a-PD-L1×4-1BB and anti-mPD-1 compared to each single agent (
Together, these results suggest that the combination of mbsIgG2a-PD-L1×4-1BB and anti-mPD-1 leads to distinct and complementary modulation of the tumor immune contexture compared to treatment with mbsIgG2a-PD-L1×4-1BB or anti-mPD-1 as single agents. In particular, the greater frequency of proliferating and cytotoxic CD8+ TILs in the mbsIgG2a-PD-L1×4-1BB with anti-PD1 combination treated group indicates enhanced functional and effector functions of TILs likely associated with improved antitumor activity.
Objective: To investigate cytokine levels in peripheral blood of MC38-tumor bearing C57BL/6 mice treated with mbsIgG2a-PD-L1×4-1BB either alone or in combination with an anti-mPD-1 antibody.
In the experiment described in Example 11, blood samples were collected from the MC38-tumor bearing C57BL/6 mice at the following time points: Day −1 (baseline; one day before treatment with the first dose), Day 2 (2 days after first dose) and Day 5 (2 days after second dose) after initiation of treatment.
Cytokines were analyzed in plasma samples by electrochemiluminescence (ECLIA) using the V-PLEX Proinflammatory Panel 1 mouse Kit (MSD LLC, cat. no. K15048D-2) and the V-PLEX Cytokine Panel 1 mouse Kit (MSD LLC, cat. no. K15245D-2) on a MESO QuickPlex SQ 120 instrument (MSD, LLC. R31QQ-3), according to the manufacturer's instructions.
In mice treated with mIgG2a-ctrl-AAKR (5 mg/kg) or anti-mouse PD-1 antibody (anti-mPD-1; 10 mg/kg) as single agent, no or minor changes in the levels of IFNγ, TNFα, IL-2 and IP-10 were observed on Day 2 or Day 5 compared to Day −1 (
These results provide rationale for evaluating the combination of GEN1046 with an anti-PD-1 antibody to further amplify the anti-tumor immune response in cancer patients.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2022/077749 | 10/5/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63257901 | Oct 2021 | US | |
| 63253106 | Oct 2021 | US |
