Patent Application
20240269232
The present invention claims priority from the European applications EP21190004.8 and EP21190005.5 filed on 5 Aug. 2021, and EP21207324.1 filed on 9 Nov. 2021.
The present invention relates to modifications to HLA-B57 polypeptides that confers increased expression from mammalian cell lines, for the purpose of producing a medicament.
Classical MHC-1 molecules (also known as HLA-I in humans) are dimeric or trimeric structures comprising a membrane-bound heavy chain with three extracellular domains (α1, α2, and α3), bound non-covalently to a β2-microglobulin (β2m) light chain, and optionally, a small peptide associated with the peptide-binding cleft. However, MHC Class I molecules may also disassociate into free heavy chains lacking β2m (Arosa et al., Trends in Immunology 2007 March; 28(3):115-23).
The inventors have previously identified the HLA-B57 heavy chain linked to certain immune correlates in HIV infection, as a promising immunomodulatory medicament (see WO 2017153438 A1). To obtain isolated HLA-B57 heavy chain fusion molecules, the HLA heavy chain must first be co-expressed with β2m. Once purified from the supernatant, the HLA heavy chain/β2m complex may be separated, and the isolated HLA chains purified and subjected to refolding. However, while β2m-non-associated HLA-B57 has desirable immunomodulatory qualities, upscaling of manufacturing to industrial quantities has revealed challenges. HLA-B57 heavy chains form oligomers and aggregates in solution, leading to reduced cell viability, and low yields of both the HLA heavy chain/β2m complex, and the derived isolated β2m-non-associated HLA heavy fusion protein.
Based on the above-mentioned state of the art, the objective of the present invention is to provide a pharmaceutically active HLA-B57 heavy chain amenable to high yield recombinant protein expression, and characterized by desirable immunomodulatory properties. This objective is attained by the subject-matter of the independent claims of the present specification, with further advantageous embodiments described in the dependent claims, examples, figures and general description of this specification.
A first aspect of the invention relates to a method to obtain an HLA-B57 fusion protein comprising a variant of the extracellular domain of an HLA-B57 heavy chain, characterized by favorable manufacturing and immunomodulatory qualities. The method comprises introducing at least one, or two amino acid substitutions into the extracellular domain of a naturally occurring HLA-B57 heavy chain polypeptide. The resulting variant HLA-B57 polypeptide is joined to a stabilizing polypeptide, to obtain a modified, variant HLA-B57 fusion protein which is highly expressed in cell culture. The key amino acid residues identified in the naturally occurring HLA-B57 polypeptide sequence by the invention, are the exchange of an alanine (A), for a glutamate (E) at position 46, and/or a valine (V) or tryptophan (W) for an arginine (R) at position 97. The positions numbers for this, and any other HLA-B57 heavy chain polypeptides referred to herein, are defined sequentially by assigning the glycine (G), serine (S), histidine (H) motif initiating the extracellular domain of the naturally occurring full-length HLA-B57 heavy-chain to the positions 1, 2 and 3, respectively.
Another aspect of the invention is an isolated HLA fusion protein, comprising a variant HLA-B57 polypeptide based on the extracellular domain of a naturally occurring HLA-B57 amino acid sequence as above, but unlike the naturally occurring sequence, characterized by an E at position 46, and an R at position 97, and linked to an IgG Fc portion.
Further aspects of the invention relate to a pharmaceutical composition comprising the isolated HLA fusion protein as specified above, or a nucleic acid, or nucleic acid expression vector encoding said HLA fusion protein, for use as a medicament for the purpose of treating a malignant neoplastic disease. Pharmaceutical compositions according to the invention, comprise at least one of the compounds of the present invention and at least one pharmaceutically acceptable carrier, diluent or excipient.
For purposes of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition set forth below conflicts with any document incorporated herein by reference, the definition set forth shall control.
The terms “comprising”, “having”, “containing”, and “including”, and other similar forms, and grammatical equivalents thereof, as used herein, are intended to be equivalent in meaning and to be open-ended in that an item or items following any one of these words is not meant to be an exhaustive listing of such item or items, or meant to be limited to only the listed item or items. For example, an article “comprising” components A, B, and C can consist of (i.e., contain only) components A, B, and C, or can contain not only components A, B, and C but also one or more other components. As such, it is intended and understood that “comprises” and similar forms thereof, and grammatical equivalents thereof, include disclosure of embodiments of “consisting essentially of” or “consisting of.”
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit, unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.
Reference to “about” a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X.”
As used herein, including in the appended claims, the singular forms “a”, “or” and “the” include plural referents unless the context clearly dictates otherwise.
“And/or” where used herein is to be taken as specific recitation of each of the two specified features or components with or without the other. Thus, the term “and/or” as used in a phrase such as “A and/or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques and biochemistry). Standard techniques are used for molecular, genetic and biochemical methods (see generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Ausubel et al., Short Protocols in Molecular Biology (2002) 5th Ed, John Wiley & Sons, Inc.) and chemical methods.
The term polypeptide in the context of the present specification relates to a molecule consisting of 50 or more amino acids that form a linear chain wherein the amino acids are connected by peptide bonds. The amino acid sequence of a polypeptide may represent the amino acid sequence of a whole (as found physiologically) protein or fragments thereof. The term “polypeptides” and “protein” are used interchangeably herein and include proteins and fragments thereof. Polypeptides are disclosed herein as amino acid residue sequences.
Amino acid residue sequences are given from amino to carboxyl terminus. Capital letters for sequence positions refer to L-amino acids in the one-letter code (Stryer, Biochemistry, 3rd ed. p. 21). Lower case letters for amino acid sequence positions refer to the corresponding D- or (2R)-amino acids. Sequences are written left to right in the direction from the amino to the carboxy terminus. In accordance with standard nomenclature, amino acid residue sequences are denominated by either a three letter or a single letter code as indicated as follows: Alanine (Ala, A), Arginine (Arg, R), Asparagine (Asn, N), Aspartic Acid (Asp, D), Cysteine (Cys, C), Glutamine (Gln, Q), Glutamic Acid (Glu, E), Glycine (Gly, G), Histidine (His, H), Isoleucine (Ile, I), Leucine (Leu, L), Lysine (Lys, K), Methionine (Met, M), Phenylalanine (Phe, F), Proline (Pro, P), Serine (Ser, S), Threonine (Thr, T), Tryptophan (Trp, W), Tyrosine (Tyr, Y), and Valine (Val, V).
The terms gene expression or expression, or alternatively the term gene product, may refer to either of, or both of, the processes—and products thereof—of generation of nucleic acids (RNA) or the generation of a peptide or polypeptide, also referred to transcription and translation, respectively, or any of the intermediate processes that regulate the processing of genetic information to yield polypeptide products. The term gene expression may also be applied to the transcription and processing of an RNA gene product, for example a regulatory RNA or a structural (e.g. ribosomal) RNA. If an expressed polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell. Expression may be assayed both on the level of transcription and translation, in other words mRNA and/or protein product.
The term nucleic acid expression vector in the context of the present specification relates to a plasmid, a viral genome, or an RNA, which is used to transfect (in case of a plasmid or an RNA) or transduce (in case of a viral genome) a target cell with a certain gene of interest, or—in the case of an RNA construct being transfected—to translate the corresponding protein of interest from a transfected mRNA. For vectors operating on the level of transcription and subsequent translation, the gene of interest is under control of a promoter sequence and the promoter sequence is operational inside the target cell, thus, the gene of interest is transcribed either constitutively or in response to a stimulus or dependent on the cell's status. In certain embodiments, the viral genome is packaged into a capsid to become a viral vector, which is able to transduce the target cell.
In the context of the present specification, the terms sequence identity and percentage of sequence identity refer to a single quantitative parameter representing the result of a sequence comparison determined by comparing two aligned sequences position by position. Methods for alignment of sequences for comparison are well-known in the art. Alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981), by the global alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Nat. Acad. Sci. 85:2444 (1988) or by computerized implementations of these algorithms, including, but not limited to: CLUSTAL, GAP, BESTFIT, BLAST, FASTA and TFASTA. Software for performing BLAST analyses is publicly available, e.g., through the National Center for Biotechnology-Information (http://blast.ncbi.nlm.nih.gov/).
One example for comparison of amino acid sequences is the BLASTP algorithm that uses the default settings: Expect threshold: 10; Word size: 3; Max matches in a query range: 0; Matrix: BLOSUM62; Gap Costs: Existence 11, Extension 1; Compositional adjustments: Conditional compositional score matrix adjustment. One such example for comparison of nucleic acid sequences is the BLASTN algorithm that uses the default settings: Expect threshold: 10; Word size: 28; Max matches in a query range: 0; Match/Mismatch Scores: 1.−2; Gap costs: Linear. Unless stated otherwise, sequence identity values provided herein refer to the value obtained using the BLAST suite of programs (Altschul et al., J. Mol. Biol. 215:403-410 (1990)) using the above identified default parameters for protein and nucleic acid comparison, respectively.
Reference to identical sequences without specification of a percentage value implies 100% identical sequences (i.e. the same sequence).
As used herein, the term pharmaceutical composition refers to an HLA fusion protein according to the invention, together with at least one pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical composition according to the invention is provided in a form suitable for topical, parenteral or injectable administration.
As used herein, the term pharmaceutically acceptable carrier includes any solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (for example, antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, and the like and combinations thereof, as would be known to those skilled in the art (see, for example, Remington: the Science and Practice of Pharmacy, ISBN 0857110624).
As used herein, the term treating or treatment of any disease or disorder (e.g. cancer) refers in one embodiment, to ameliorating the disease or disorder (e.g. slowing or arresting or reducing the development of the disease or at least one of the clinical symptoms thereof). In another embodiment “treating” or “treatment” refers to alleviating or ameliorating at least one physical parameter including those which may not be discernible by the patient. In yet another embodiment, “treating” or “treatment” refers to modulating the disease or disorder, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter), or both. Methods for assessing treatment and/or prevention of disease are generally known in the art, unless specifically described hereinbelow.
In the context of the present specification, the term peptide linker, or amino acid linker refers to a polypeptide of variable length that is used to connect two polypeptides in order to generate a single chain polypeptide. Exemplary embodiments of linkers useful for practicing the invention specified herein are oligopeptide chains consisting of 1, 2, 3, 4, 5, 10, 20, 30, 40 or 50 amino acids. A non-limiting example of an amino acid linker is the flexible, glycine-rich peptide GGGGSGGGGS (SEQ ID NO 003) used to link a variant HLA-B57 polypeptide with a stabilizing peptide to provide an HLA fusion protein in the examples.
The term human leukocyte antigen, HLA, HLA heavy chain, or HLA alpha chain in the context of the present specification relates to the family of proteins encoded by the Class/Major histocompatibility (MHC) antigen gene family on chromosome 6. The HLA heavy chain can be a monomer, or form a part of dimeric structures comprising a heavy chain with three extracellular domains (α1, α2, and α3), bound non-covalently to a β2m light chain, or optionally, trimeric structures wherein a small peptide epitope is associated at the peptide-binding cleft.
The term or naturally occurring HLA-B57 (HLA-B*57, B57, HLA-B57, HLA-B57 polypeptide) refers specifically to the products of the MHC Class I HLA-B57 subfamily of genes, encoding numerous similar proteins. The HLA-B57 family currently encompasses 221 known alleles with unique nucleic acid sequences, and several dozen unique protein sequences, numbering HLA-B*57:01 to HLA-B*57:141 (the sequence for which are known, and may be retrieved, for example, by entering the search term “B*57” into the MGT/HLA Allele Query Form provided by the European Bioinformatics Institute Immuno Polymorphism Database, Robinson J. et al. 2013 Nucleic Acids Res. 41:D1234, https://www.ebi.ac.uk/ipd/imgt/hla/allele.html). Full length naturally occurring HLA-B57 comprise an extracellular domain comprising an α1, an α2, and an α3 domain, a connecting peptide region, a transmembrane domain, and an intracellular domain. The term variant in the context of the present specification relating to an HLA heavy chain polypeptide sequence, refers to a polypeptide with at least one amino acid residue that differs from a naturally-occurring HLA polypeptide sequence. For example, a variant HLA heavy chain polypeptide in which one, or several amino acid substitutions have been introduced, such that it differs from the original, naturally occurring human protein sequence it is derived from.
The term extracellular domain as applied to an HLA-B57 polypeptide, or variant HLA-B57 polypeptide in the context of the specification refers to the extracellular portion of the HLA-B57 heavy chain protein, comprising at least an α1, an α2, and an α3 domain.
In the context of the present specification, the terms HLA fusion protein or HLA-B57 fusion protein refer to a polypeptide which comprises, or essentially consists of a wildtype or a variant HLA-B57 extracellular domain polypeptide, joined to a stabilizing Fc polypeptide domain, optionally by means of a peptide linker. An HLA fusion protein according to the invention, comprising the variant HLA-B57 polypeptide as specified in the present description, is sometimes referred to in the examples herein as HLA-B57(A46E, V97R). The term encompasses both an HLA fusion protein in complex with a β2-microglobulin polypeptide secreted from a cell culture, and the purified, HLA fusion protein which is has been separated from β2-microglobulin. The term HLA fusion protein encompasses both a monomer form, comprising a single HLA-B57 polypeptide joined to a stabilizing immunoglobulin (Ig) Fc domain, or a dimer formed by association of a first HLA fusion protein monomer, and a second HLA fusion protein monomer, for example, via association of Ig Fc domains.
In the context of the present specification, the term β2-microglobulin protein (β2m protein, β2m, B2m, B2M, HLA light chain) refers to the beta (β) chain, also known as the light chain, of the MHC class I heterodimer. The term β2-microglobulin protein encompasses firstly, a pre-processing β2-microglobulin protein comprising a secretory signal, for example, the sequence of Uniprot P61769, or the sequence SEQ ID NO 014, and secondly, the post-secretion form of the protein, in which a secretory signal portion of the protein has been removed by cleavage as the polypeptides are exported from the cell.
In the context of the present specification, the term β2m-non-associated HLA heavy chain refers to an HLA heavy chain molecule, particularly an HLA fusion protein according to the invention, lacking association with a β2m molecule. To obtain an HLA fusion protein β2m-non-associated HLA heavy chain by means of the recombinant protein manufacturing processes used herein, a molecule comprising an HLA heavy chain polypeptide is expressed together with a β2m protein in a mammalian cell, and the resulting HLA: β2m complex. The presence of β2m is sometimes indicated by the suffix “0.52m” following a compound. β2m is then separated under acidic conditions, after which the β2m-free HLA fusion protein is purified by size exclusion. In the context of the present specification, the terms HLA-B57.no β2m or HLA-B57(A46E/V97R).no β2m refer to HLA fusion proteins comprising the non-variant, and variant HLA heavy chain polypeptide linked to an IgG Fc polypeptide respectively (in which (A46E/V97R) denotes the presence of the variant HLA polypeptide according to the invention), which has been separated from the β2m protein.
In the context of the present specification, the term secretory signal, secretory signal peptide or signal sequence refers to an N-terminal leader sequence initiating the open reading frame (ORF) of a polypeptide, usually about 6-30 amino acids in length. In rare cases, a secretory signal is placed at the C-terminus of a polypeptide. Secretory signals are sometime referred to as targeting signals, localization signals, transit peptides, leader sequences, or leader peptides. Secretory signals which enable efficient secretion of a polypeptide from a cell are well known in the art, and may be included in the ORF of a recombinant protein in order to facilitate export of a polypeptide to the supernatant in cell-based polypeptide manufacturing system, allowing purification of a polypeptide from the cell supernatant. Upon translation of the mRNA encoding the secretory signal, it is recognized by a cytosolic protein mediating transfer of the mRNA-ribosome complex to a channel protein in the endoplasmic reticulum (ER). The newly synthesized polypeptide comprising the secretory signal peptide is translocated to the ER lumen through the channel protein, entering the cell secretion pathway. Signal sequences of particular use according to the invention are those that are cleaved from the final polypeptide product following translation, such as those used in the examples such as SEQ ID NO 019, or that comprised within SEQ ID NO 020.
In the context of the present specification, the term antibody refers to whole antibodies including but not limited to immunoglobulin type G (IgG), type A (IgA), type D (IgD), type E (IgE) or type M (IgM), any antigen-binding fragment or single chains thereof and related or derived constructs. A whole antibody is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region of IgG is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region (CL). The light chain constant region is comprised of one domain, CL. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. 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 the first component of the classical complement system. Similarly, the term encompasses a so-called nanobody or single domain antibody, an antibody fragment consisting of a single monomeric variable antibody domain.
In the context of the present specification, the term immunoglobulin crystallizable fragment (Fc) region, or Ig Fc refers to a fraction of an antibody, or immunoglobulin (Ig), comprised of a CH2 and a CH3 domain. Ig Fc encompasses both a monomer, or a dimer comprising two Ig Fc, covalently linked by disulfide bonds. In the context of the HLA fusion protein according to the invention, disulfide bonds can join two HLA fusion proteins molecules, each comprising Ig Fc domains. The presence of the Ig Fc in the HLA fusion protein facilitates increased solubility, stability, avidity, half-life, and from a technological point of view, cost-effective production and purification in mammalian systems (protein A or G purification).
The term antibody-like molecule in the context of the present specification refers to a molecule capable of specific binding to another molecule or target with high affinity/a Kd≤10E−8 mol/l. An antibody-like molecule binds to its target similarly to the specific binding of an antibody. The term antibody-like molecule encompasses a repeat protein, such as a designed ankyrin repeat protein (Molecular Partners, Zurich), an engineered antibody mimetic protein exhibiting highly specific and high-affinity target protein binding (see US2012142611, US2016250341, US2016075767 and US2015368302, all of which are incorporated herein by reference). The term antibody-like molecule further encompasses, but is not limited to, a polypeptide derived from armadillo repeat proteins, a polypeptide derived from leucine-rich repeat proteins and a polypeptide derived from tetratricopeptide repeat proteins. The term antibody-like molecule further encompasses a specifically binding polypeptide derived from a protein A domain, a fibronectin domain FN3, a consensus fibronectin domain, a lipocalin (see Skerra, Biochim. Biophys. Acta 2000, 1482(1-2):337-50), a polypeptide derived from a Zinc finger protein (see Kwan et al. Structure 2003, 11(7):803-813), a Src homology domain 2 (SH2) or Src homology domain 3 (SH3), a PDZ domain, a gamma-crystallin, ubiquitin, a cysteine knot polypeptide or a knottin, cystatin, Sac7d, a triple helix coiled coil (also known as alphabodies), a Kunitz domain or a Kunitz-type protease inhibitor and a carbohydrate binding module 32-2.
The term armadillo repeat protein refers to a polypeptide comprising at least one armadillo repeat, wherein an armadillo repeat is characterized by a pair of alpha helices that form a hairpin structure.
The term humanized camelid antibody in the context of the present specification refers to an antibody consisting of only the heavy chain or the variable domain of the heavy chain (VHH domain) and whose amino acid sequence has been modified to increase their similarity to antibodies naturally produced in humans and, thus show a reduced immunogenicity when administered to a human being. A general strategy to humanize camelid antibodies is shown in Vincke et al. “General strategy to humanize a camelid single-domain antibody and identification of a universal humanized nanobody scaffold”, J Biol Chem. 2009 Jan. 30; 284(5):3273-3284, and US2011165621A1.
In the context of the present specification, the term checkpoint inhibitory agent encompasses a cancer immunotherapy agent capable of disrupting an inhibitory signaling cascade that limits immune cell activation, and in particular T cell activation, known in the art as an immune checkpoint mechanism, particularly a checkpoint inhibitor antibody or an antibody-like molecule, such as an antibody fragment, a diabody, or a single variable chain antibody fragment. Examples of checkpoint inhibitory agents include, for example, an antibody, antibody like molecule, or natural ligand receptor which binds specifically to CTLA-4 (Uniprot P16410), PD-1 (Uniprot Q15116), PD-L1 (Uniprot Q9NZQ7), B7H3 (CD276; Uniprot Q5ZPR3), VISTA (Uniprot Q9H7M9), TIGIT (UniprotQ495A1), or TIM-3 (HAVCR2, Uniprot Q8TDQ0), CD137 (41BB, Uniprot Q07011), CD40 (Uniprot Q09LL4), CD27 (Uniprot P26841), OX40 (CD134, UniprotP43489), NKG2A (Uniprot P26715), CD86 (Uniprot P42081), CD80 (Uniprot P33681), or LAG-3 (Uniprot P18627).
The terms “cancer” and “malignant neoplastic disease” are used synonymously herein. Particular alternatives of any of the aspects and embodiments disclosed herein are directed at the use of the combinations of the invention in treatment of solid tumours. Other alternatives of any of the aspects and embodiments disclosed herein are directed at the use of the combinations of the invention in treatment of liquid cancers such as myelogenous or granulocytic leukemia, particularly AML, lymphatic, lymphocytic, or lymphoblastic leukemia and lymphoma, polycythemia vera or erythremia.
A first aspect of the invention relates to a method for producing an HLA-B57 fusion protein with improved mammalian cell system expression properties, said HLA-B57 fusion protein comprising a variant HLA-B57 polypeptide, joined to a stabilizing polypeptide. The method involves altering specific amino acids in the extracellular domain of a naturally occurring HLA-B57 heavy-chain polypeptide, to provide a variant which retains desirable immunomodulatory qualities, but is easier to manufacture as a result of enhanced stability, less propensity to form aggregates in solution, and higher expression titers.
In some embodiments, the method according to this aspect of the invention comprises introducing into a cell, a nucleic acid sequence encoding a polypeptide comprising a variant of the extracellular domain of a naturally occurring HLA-B57 heavy-chain, fused to a stabilizing polypeptide, wherein the variant HLA-B57 heavy chain polypeptide (but not the naturally occurring HLA-B57 heavy-chain polypeptide it is derived from) is characterized by a glutamate (E) at position 46. The amino acid position numbering according to the invention is defined sequentially by assigning the initial glycine (G), serine (S), histidine (H) motif of the extracellular domain of a naturally occurring HLA-B57 heavy-chain to positions 1, 2 and 3, respectively. In other words, the secretory signal peptide is not included in the numbering.
In some embodiments, the method according to this aspect of the invention, comprises introducing into a cell a nucleic acid sequence encoding an HLA fusion protein comprising a variant of the extracellular domain of a naturally occurring HLA-B57 heavy-chain fused to a stabilizing polypeptide. According to this embodiment, the variant HLA-B57 heavy chain polypeptide (but not the naturally occurring HLA-B57 heavy-chain polypeptide it is derived from) is characterized by an arginine (R) at position 97.
In certain embodiments of the method according to the invention, the nucleic acid sequence encoding the HLA fusion protein encodes a variant HLA-B57 polypeptide which is identical to the extracellular portion of any naturally occurring HLA-B57 heavy chain polypeptide which:
Another aspect of the invention is a method for producing an HLA fusion protein comprising as a first step, introducing amino acid substitutions into the extracellular domain of any naturally occurring, full length HLA-B57 heavy-chain polypeptide, to provide a variant HLA-B57 polypeptide characterized by an E at position 46, and an R at position 97. Depending on the HLA-B57 sequence, this may entail introducing an amino acid substitution encoding an E at position 46 (A46E), and/or introducing an amino acid substitution encoding an R at position 97 (V97R or W97R). The method according to this aspect of the invention comprises a second step, introducing into a cell, a nucleic acid sequence encoding an HLA fusion protein comprising said variant HLA-B57 polypeptide joined to a stabilizing polypeptide.
The method to produce an HLA fusion protein according to the first, or the second aspect of the invention, comprises introducing into the same cell, a nucleic acid encoding a β2m polypeptide sequence, to increase the expression level, and secretion of the HLA fusion protein in the form an HLA heavy-chain/β2m complex, also referred to as HLA.β2m. HLA.β2m can be further dissociated to provide isolated HLA molecules lacking β2m. The nucleic acids encoding the HLA fusion protein, and the β2m polypeptide, according to these aspects of the invention, are under control of a promoter sequence operable in said cell. The cell is then cultured under conditions where the HLA-B57 and the β2m encoding nucleic acid sequences are expressed, to provide the HLA.β2m complex. In particular embodiments of the method according to the invention, the HLA fusion protein is associated with a β2m molecule at a molar ratio of between 3:5 to 7:5, more particularly between 4:5 to 6:5. In other words, the HLA fusion protein and the β2m polypeptide are present at a ratio of, or close to, 1 to 1.
In particular embodiments of the first or second aspect of the invention, the stabilizing polypeptide joined to the variant HLA-B57 polypeptide, is an Ig Fc polypeptide. In more particular embodiments, the stabilizing peptide is an IgG Fc polypeptide. In still more particular embodiments, the stabilizing peptide is an IgG4 Fc polypeptide. In even more particular embodiments, the stabilizing peptide is an IgG4 Fc polypeptide with the sequence SEQ ID NO 004. In other embodiments which the inventors consider feasible, the stabilizing polypeptide is an albumin polypeptide, such as bovine, or human serum albumin. In other embodiments considered feasible by the inventors, the stabilizing polypeptide is a PEG-containing compound.
In particular embodiments of the methods to produce an HLA heavy chain fusion protein according to the invention, the obtained HLA fusion protein comprises, from the N′ to the C′ terminus of the fusion protein:
In certain embodiments of the methods to produce an HLA heavy chain fusion protein according to the invention, the HLA fusion protein nucleic acid sequence, and the β2m nucleic acid sequence, are present on a single nucleic acid vector molecule (for example, on a single plasmid). In particular embodiments, the HLA fusion protein nucleic acid sequence, and the β2m nucleic acid sequence are present on different nucleic acid vector molecules (for example, expressed from two different plasmids). In such embodiments, the optimal ratio of the nucleic acid vector comprising the HLA fusion protein nucleic acid sequence with respect to the nucleic acid vector comprising the β2m nucleic acid sequence is ≥1 and ≤2, particularly the molar ratio is >1 and <2, as demonstrated in
In some embodiments of the methods to produce an HLA fusion protein according to the invention as specified above, the method comprises the following additional steps:
Step a. will provide an HLA fusion protein associated with β2m protein, and step b. an HLA fusion protein disassociated from β2m protein—the B2m-free polypeptide demonstrated to antagonize tumor growth when administered in vivo in
In some embodiments of the methods for producing an isolated HLA fusion protein according to either the first or second aspect of the invention, a further dissociation step is carried out to provide a β2m-non-associated conformer. This may be achieved, for example, by exposure of HLA fusion protein: β2m protein complex to acidic conditions, particularly approximately pH 3, allowing separation of β2m-non-associated conformer HLA fusion protein from the β2m protein by size exclusion chromatography. In some embodiments of the methods, the method may further comprise a desalting step, wherein following dissociation of β2m from the HLA fusion protein/β2m protein complex, the purified HLA fusion proteins are brought to a physiological pH, allowing correct folding of the protein.
In certain embodiments of the methods to produce an HLA fusion protein according to either the first or second aspect of the invention, the naturally occurring extracellular domain of an HLA-B57 heavy-chain polypeptide forming the base sequence for the variant HLA-B57 polypeptide, is characterized by an A at position 46. This embodiment encompasses the use of any HLA-B57 heavy chain as a starting point for creating the variant HLA heavy chain polypeptide, except those where position 46 is already E (for example, HLA-B57:16, HLA-B57:45, HLA-B57:51, or HLA-B57:69). In other words, in order to obtain the variant sequence from the naturally occurring sequence, at least one (A46E), and possibly two amino acid substitutions (both A46E and either V97R, or W97R) are introduced at the indicated sites of the naturally-occurring HLA-B57 extracellular domain.
In alternative embodiments of the methods to produce an HLA fusion protein according to either the first or second aspect of the invention, the extracellular domain of the naturally occurring HLA-B57 heavy-chain is characterized by a V at position 97. This encompasses any HLA-B57 heavy chain except those where position 97 is already R (for example, HLA-B57:05, HLA-B57:82, HLA-B57:83, HLA-B57:118, or HLA-B57:131), or where position 97 is a W (HLA-B57:11). In these embodiments, in order to obtain the variant sequence from the naturally occurring sequence, at least one (V97R), and possibly two amino acid substitutions (both V97R and A46E) are introduced at the indicated sites.
In other embodiments of the methods to produce an HLA fusion protein according to either the first or second aspect of the invention, the extracellular domain of the naturally occurring HLA-B57 heavy-chain is characterized by an A at position 46, and either a V, or a W at position 97. In other words, the naturally occurring HLA-B57 haplotype may be any HLA-B57 heavy chain except those where position 46 is already an E (for example, HLA-B57:16, HLA-B57:45, HLA-B57:51, HLA-B57:69) or position 97 is already an R (for example, HLA-B57:05, HLA-HLA-B57:82, HLA-B57:83, HLA-B57:118, or HLA-B57:131). In these embodiments, in order to obtain the variant sequence from the naturally occurring sequence, two amino acid substitutions, an A46E and either a V97R or W97R, are introduced at the indicated sites to provide the variant sequence.
In particular embodiments of the methods to produce an HLA fusion protein according to the invention, two amino acid substitutions A46E, and V97R are introduced into a naturally occurring polypeptide sequence of any HLA-B57 extracellular domain which is characterized by both an A at position 46, and a V at position 97. This embodiment encompasses the use of any HLA-B57 haplotype as a starting point for obtaining the variant HLA polypeptide domain of the fusion protein, except those where position 46 is already an E (for example, HLA-B57:16, HLA-B57:45, HLA-B57:51, or HLA-B57:69), or where position 97 is already R (HLA-B57:05, HLA-B57:82, HLA-B57:83, HLA-B57:118, HLA-B57:131), or where position 97 is a W (HLA-B57:11).
In further particular embodiments of the methods to produce an HLA fusion protein according to either the first or second aspect of the invention, the variant HLA-B57 polypeptide portion of the HLA fusion protein comprises the sequence SEQ ID NO 002. In other words, the method to produce an HLA fusion protein according to this embodiment involves introducing into a cell a nucleic acid encoding the β2m protein, and a nucleic acid encoding an HLA fusion protein (comprising a variant HLA heavy chain polypeptide joined to an IgG4 Fc molecule), where the variant HLA-B57 polypeptide is identical to the naturally occurring HLA-B57:01 sequence SEQ ID NO 001, apart from amino acid substitutions that encode an E at position 46, and an R at position 97. This cell is then cultured in conditions amenable to expression of both proteins, to provide an HLA fusion protein/β2m protein complex.
In particular embodiments of the methods to obtain an HLA fusion protein according to either the first or second aspect of the invention, the nucleic acid sequence encoding an HLA fusion protein, encodes a polypeptide sequence comprising the sequence SEQ ID NO 015, the fusion protein as it is found following secretion from mammalian cells, lacking a secretion signal. In more particular embodiments of the methods to obtain an HLA fusion protein, the nucleic acid sequence encodes an HLA fusion protein polypeptide sequence which comprises the sequence SEQ ID NO 005, or SEQ ID NO 020, where the HLA fusion polypeptide sequence is preceded by one of two secretory signals the inventors found to facilitate efficient secretion of the HLA fusion protein from the cell. In still more particular embodiments of the method, the nucleic acid encodes an HLA polypeptide which essentially consists of SEQ ID NO 005.
In some embodiments of the methods to obtain an HLA fusion protein, the nucleic acid sequence encoding an HLA fusion protein comprises the sequence SEQ ID NO 016, preferably associated with an additional nucleic acid encoding a secretion signal sequence.
In further particular embodiments of the methods to produce an HLA heavy chain fusion protein, the HLA fusion protein nucleic acid sequence encodes a secretory signal. In still more particular embodiments, it encodes a secretory signal 16 to 30 amino acids in length, positioned N-terminal to the encoded variant HLA-B57 polypeptide sequence. In still more particular embodiments, the secretory signal encoded by HLA fusion protein nucleic acid sequence has the sequence SEQ ID NO 019.
In particular embodiments of the methods to obtain an HLA fusion protein, the nucleic acid sequence encoding an HLA fusion protein comprises the sequence SEQ ID NO 006, which encodes a useful secretion signal preceding the HLA fusion protein. In more particular embodiments of the methods to obtain an HLA fusion protein, the nucleic acid sequence encoding an HLA fusion protein essentially consists of the sequence SEQ ID NO 006. The secretory signal encoded by the HLA fusion protein nucleic acid sequence is removed by cleavage during the process of secretion from the cell.
In some embodiments of any one of the aspects of the method to obtain an HLA fusion protein specified above, the cell producing the HLA fusion protein is a eukaryotic cell. In particular embodiments, the cell is a mammalian cell. In more particular embodiments of the method to produce an HLA fusion protein as demonstrated in the examples, a Chinese hamster ovarian (CHO) cell is used to express the recombinant fusion protein product, though the inventors predict other cell types such as insect cells, or particularly other mammalian cell types, can feasibly produce an HLA fusion protein according to the invention.
A next aspect of the invention is an isolated HLA fusion protein comprising firstly, a variant HLA-B57 polypeptide which differs by at least 1, or 2 amino acids from the extracellular domain (as specified according to the definition of the term variant HLA-B57 polypeptide) of a naturally occurring HLA-B57 heavy-chain polypeptide. Said variant HLA-B57 polypeptide is characterized by an E at position 46, and an R at position 97, wherein the amino acids residues are numbered from the G,S,H motif located at the N-terminal region of extracellular alpha 1 domain (using amino acid numbering as specified in the definition of the term variant HLA-B57 polypeptide). Secondly, the isolated HLA fusion protein comprises a stabilizing polypeptide, for example, an Ig Fc. In particular embodiments, the stabilizing polypeptide is an IgG Fc. In even more particular embodiments, the stabilizing peptide is an IgG4 Fc. In even more particular embodiments, the stabilizing peptide is an IgG4 Fc with desirable pharmaceutical qualities, with the polypeptide sequence designated SEQ ID NO 004.
In some embodiments of an isolated HLA fusion protein according to the invention, it comprises a variant HLA-B57 polypeptide obtained by introducing amino acid substitutions according to the method according to any one of the aspects or embodiments specified in the section Improved methods for production of an HLA-B57 fusion protein, into the extracellular portion of a naturally occurring HLA-B57 polypeptide, such that it is characterized by an E at position 46 and an R at position 97.
In particular embodiments, the isolated HLA fusion protein of the invention comprises a variant HLA-B57 polypeptide comprising the sequence SEQ ID NO 002. In more particular embodiments, the isolated HLA fusion protein of the invention comprises a variant HLA-B57 polypeptide essentially consisting of the sequence designated SEQ ID NO 002.
In further particular embodiments of the isolated HLA-B57 fusion protein according to the invention, it essentially consists of a variant HLA-B57 polypeptide and a stabilizing polypeptide joined by a peptide linker. In particular embodiments, the peptide linker is between 5 and 20 amino acids in length. In more particular embodiments this joining peptide linker has the sequence SEQ ID NO 003.
In still further particular embodiments, the isolated HLA fusion protein comprises the sequence designated SEQ ID NO 015. In still more particular embodiments, the isolated HLA fusion protein essentially consists of a polypeptide with the sequence designated SEQ ID NO 015.
In certain embodiments of the isolated HLA fusion protein, it is associated with a β2m molecule at a molar ratio of between 3:5 to 7:5, more particularly between 4:5 to 6:5. In other words, the HLA fusion protein and the β2m polypeptide are present at a ratio of, or close to, 1 to 1.
All naturally occurring HLA-B57 heavy chains comprising a full-length extracellular domain, are characterized by either an alanine (A) at position 46, and/or a valine (V) or tryptophan (W) at position 97. The sequences of the HLA-B57 family of proteins are retrievable by entering the search term “B*57” into the Alleles Resource of the Immuno polymorphism database (IBD, https://www.ebi.ac.uk/ipd/imgt/hla/allele.html, Robinson J. et al. 2013 Nucleic Acids Res. 41:D1234). A majority are characterized by an A at position 46, and a V or W at position 97. Therefore, the method according to the first and second aspect of the invention may comprise introducing either one, or two amino acid substitutions into the original protein sequence to provide a variant HLA-B57 polypeptide. In the figures presented in the examples, the manufacture of such an HLA fusion protein according to the invention, first as a complex of a variant HLA-B57 polypeptide fused to an IgG4 Fc in complex with β2m protein, and as a β2m-non-associated conformer variant HLA-B57 polypeptide IgG4 Fc fusion protein disassociated from β2m protein, is improved by amino acid substitutions at position E46 and R97, when compared to an otherwise identical structures with an unmodified HLA-B*57:01 sequence.
Certain domains of the naturally occurring HLA-B57 heavy chain not required for cognate ligand interactions, the intracellular domain, and the transmembrane domain, are absent from the HLA fusion protein according to any one of the aspects of the invention herein. In particular embodiments of the method to produce an HLA fusion protein, or the isolated HLA fusion protein according to the invention, the variant HLA-B57 polypeptide comprises the core structure of the extracellular portion of the naturally occurring HLA heavy chain protein sequence, comprising the alpha 1, 2, and 3 domains, as this portion confers the fusion protein with the ability to interact with surface molecules on target cells.
In particular embodiments of the methods to produce an HLA fusion protein, or the isolated HLA fusion protein according to the invention, the variant HLA-B57 polypeptide comprised in the HLA fusion protein includes the alpha 1, 2 and 3 domains of the naturally occurring HLA heavy chain protein, regions of which are essential for receptor ligand interactions which mediate the immunomodulatory effects of an HLA fusion protein according to the invention.
In more particular embodiments of the method to produce an HLA fusion protein, or the isolated HLA fusion protein according to the invention, the variant HLA-B57 polypeptide protein is the alpha 1, 2 and 3 domains of the naturally occurring HLA heavy chain protein, excepting the C-terminal isoleucine-valine dipeptide, preceded by the threonine-valine-proline residues of the extracellular domain, within the HLA-B57 region preceding the transmembrane domain sometimes annotated, or referred to, as the “connecting peptide”.
Structural data suggests that HLA-B57 interacts with ligands such as Killer immunoglobulin-like receptors (KIR) and leukocyte immunoglobulin-like receptors (LILR) via regions distant from the transmembrane region. Amino acids close to the membrane do not generally interact with receptors. Furthermore, the inventors surmise that the high content of hydrophobic amino acids within the 5 C-terminal amino of the extracellular domain of naturally occurring HLA heavy chain sequences specified in the preceding paragraph, is likely to introduce undesirable properties into recombinant fusion proteins, such as a tendency towards protein aggregation, which can then affect the production, purification, stability and toxicity in downstream production processes.
In particular embodiments of the methods to produce an HLA fusion protein, or the isolated HLA fusion protein according to the invention, the variant HLA-B57 heavy chain polypeptide is derived from an extracellular domain polypeptide of any HLA-B57 heavy chain from HLA-B*57:01 to HLA-B*57:141 (with the exception of truncated alleles lacking the full extracellular domain sequence). Amino acid substitutions in the naturally occurring polypeptide, resulting in position 46 (counting from the G,S,H motif) of the extracellular domain as an E, and the amino acid at position 97 as an R, provide a variant HLA heavy chain polypeptide, or fusion protein according to the invention. In addition to SEQ ID NO 001, suitable naturally occurring HLA-B57 heavy chain protein sequences comprising the required alpha 1, 2, and 3 domain of the extracellular portion of the polypeptide as specified in the definition of the term variant HLA-B57 polypeptide, that may serve as a starting point for constructing the variant HLA-B57 polypeptide, or HLA fusion protein according to the invention can be found in public sequence repositories. These include sequences selected from, but not limited to, the HLA-B57 heavy chains in the first column of the following table, which may be retrieved from the EMBL-EBI laboratory's IPD database (as cited in the definition of the term HLA-B57 in the Terms and Definitions section):
In other embodiments of the methods to produce an HLA fusion protein, or the isolated HLA fusion protein according to the invention, the variant HLA-B57 polypeptide is identical to the extracellular domain of any naturally occurring HLA-B57 heavy chain polypeptide which comprises a full length extracellular domain, truncated before the “connecting peptide” region, the region of the HLA-B57 heavy-chain following the extracellular domain, and preceding the transmembrane domain, apart from being characterized by an E at position 46, and an R at position 97.
In certain embodiments, the variant HLA-B57 polypeptides or variant HLA-B57 fusion proteins according to the invention may further comprise a secretory signal, for example, the initial 24 amino acids present in most naturally occurring HLA-B57 polypeptides annotated as a signal peptide, or an alternative secretion signal designed for efficient HLA fusion protein secretion, such as SEQ ID NO 019.
Additionally, in certain embodiments of the variant HLA-B57 polypeptides or variant HLA-B57 fusion proteins according to the invention, C-terminal to the alpha 1, alpha 2 and alpha 3 domains of the extracellular domain of natural HLA-B57 protein sequence, the initial 6 residues of the HLA-B57 “connecting peptide” region joining the alpha 3 domain with the transmembrane domain, may be present, which are found in most naturally occurring full-length variants of the HLA-B57 polypeptide.
A variant HLA-B57 polypeptide according to the invention can be obtained by introducing amino acid modifications into the extracellular domain of a natural HLA-B57 polypeptide sequence as specified above. Said variant HLA-B57 polypeptide in the terminology used in the current specification consists of, or comprises a variant of the portion of the extracellular domain of a natural HLA-B57 polypeptide sequence required for interaction with HLA-B57 ligands, particularly comprising at least the alpha 1, alpha 2 and alpha 3 domains of a naturally occurring HLA-B57 protein sequence.
A variant HLA-B57 polypeptide according to the invention is further characterized by:
The amino acid numbering for HLA-B57 used herein is defined sequentially by assigning the initial G,S,H motif of an extracellular domain of the variant, or naturally occurring HLA-B57 heavy-chain with the sequence SEQ ID NO 001 to the positions 1, 2 and 3, respectively.
In certain embodiments, the variant HLA-B57 polypeptide according to the invention optionally further comprises:
A variant HLA-B57 polypeptide according to the invention lacks, or its nucleic acid sequence does not encode:
In particular embodiments, the variant HLA-B57 polypeptide lacks, or its nucleic acid sequence does not encode the last 3 to 11 residues, particularly the last 5 residues of the “connecting peptide” region of the extracellular domain present in most full length naturally HLA-B57 protein sequences joining the alpha 3 domain, and the transmembrane domain.
In particular embodiments of the methods to produce an HLA fusion protein, or the isolated HLA fusion protein according to the invention, the variant HLA-B57 polypeptide is identical to the extracellular domain of any naturally occurring HLA-B57 heavy chain polypeptide which comprises a full length extracellular domain, truncated before the least 3, 4, 5, or 6 amino acid residues of the “connecting peptide” region, in addition to being characterized by an E at position 46, and an R at position 97.
In more particular embodiments of the methods to produce an HLA fusion protein, or the isolated HLA fusion protein according to the invention, the variant HLA-B57 heavy-chain polypeptide is identical to the extracellular domain of any naturally occurring HLA-B57 heavy chain polypeptide which comprises a full length extracellular domain, but lacks the 5 C-terminal amino acid residues of the “connecting peptide” region, in addition to being characterized by an E at position 46, and an R at position 97.
In particular embodiments, the variant HLA-B57 polypeptide is identical to the first 280 amino acids encoding the extracellular portion of any naturally occurring HLA-B57 heavy chain polypeptide, counting from the GSH motif of the alpha 1 domain of the extracellular domain, apart from being characterized by an E at position 46, and an R at position 97.
In alternative embodiments of the isolated HLA fusion protein according to the invention, the variant HLA-B57 polypeptide comprises a sequence identical to the extracellular domain of a variant of a naturally occurring HLA-B57 polypeptide characterized by an E at position 46, and an R at position 97, for example, SEQ ID NO 002, other than a single differing amino acid residue (wherein the single differing amino acid residue is not position 46, or 97). In other words, in addition to the one, or in some cases, two amino acid substitutions at positions 46, and/or 97, the variant HLA-B57 polypeptide according to this embodiment includes at least one further amino acid substitution compared to the naturally occurring HLA-B57 molecule from which it is derived. According to this embodiment, the resulting HLA fusion protein has at least a similar, or improved biological function to all previous embodiments of the isolated HLA fusion protein according to the invention. Specifically, the function of the HLA fusion protein can be characterized by means and methods demonstrated in the examples herein, namely in comparison to a similar construct lacking the amino acid substitutions at positions 46, and 97 (characterized by an A46, and a V97/W97). The HLA fusion protein is characterized by at least a two-fold increase in recombinant HLA fusion protein titer measured in the supernatant of a mammalian cell (as summarized in
The data in
In particular embodiments of the method for producing an HLA fusion protein, or the isolated HLA according to the invention, a β2m-non-associated conformer derived from the variant HLA fusion protein has an improved binding to LILRB2 (UniProt Q8N423) compared to an equivalent compound comprising the naturally-occurring HLA heavy chain peptide from which it is derived. In other words, binding saturation of LILRB2 with an HLA-B57(A46E, V97R) fusion protein can be achieved at a lower concentration compared an equivalent construct comprising the naturally-occurring HLA heavy chain peptide from which it is derived. LILRB2 binding saturation according to the invention particularly refers to measurements using an enzyme-linked immunoassay, where HLA fusion protein is titrated onto immobilized biotinylated LILRB2, and detected with an anti-Ig secondary antibody. In other particular embodiments, said variant HLA heavy chain incorporated into an β2m-non-associated conformer HLA fusion protein binds to LILRB2 receptor with an EC50 of equal to, or less than (≤) about 21 nM, particularly ≤about 15 nM, more particularly ≤about 10 nM, indicating more than about a 2-fold increase in LILRB2 binding saturation.
The HLA fusion protein according to any aspect of the methods, or the isolated HLA fusion protein according to the invention specified above, comprises an additional polypeptide conferring stability during expression and purification to the HLA-B57 portion of the fusion protein. The presence of stabilizing portion of the HLA fusion protein increases the yield and solubility by reducing degradation and oligomerization of the HLA fusion protein, as well as increasing viability of the cell expressing the fusion protein.
In particular embodiments of the method to produce an HLA fusion protein, or the isolated HLA fusion protein, the stabilizing polypeptide of the HLA fusion protein is a human Ig Fc polypeptide. In particular embodiments, the stabilizing peptide of the HLA fusion protein is an isotype IgG Fc. An IgG Fc stabilizing peptide domain delivers an added advantage during purification of the HLA fusion protein, by enabling absorption to a protein A or G coated surface. The Fc portion may also prolong the in vivo half-life of a molecule in vivo. The inventors consider a possible alternative that may provide similar benefits if attached to the functional HLA polypeptides in lieu of Ig Fc could be bovine serum albumin. Previous work by the inventors has established that an albumin molecule, such as bovine serum albumin, may also serve as a stabilizing polypeptide. It is also known that PEGylation can enhance the half-life of proteins in circulation, and is thus another feasible stabilizing polypeptide.
In particular embodiments of the method to produce an HLA fusion protein, or the isolated HLA fusion protein according to the invention, the HLA fusion protein comprises an IgG4 polypeptide. In more particular embodiments, the HLA fusion protein comprises an altered IgG4 S228P.dk molecule with the sequence SEQ ID NO 004. This is characterized by a mutation in the hinge region of the IgG4, where Proline (P) is substituted for serine (S) at position 228 of the original IgG4 antibody, and dK indicates a deletion of the last amino acid Lysine (K) on the original IgG4 sequence. These changes give the IgG4 format stability and less heterogenicity. Both changes are well established and used commonly in diverse Fc constructs.
In certain embodiments, the isolated HLA fusion protein is a dimer which comprises a first monomer and a second monomer, and each monomer independently of the other monomer comprises an HLA heavy chain polypeptide fused to an Ig Fc portion, the latter of which may associate via disulfide bonds.
In certain embodiments of the method to produce an HLA fusion protein, the variant HLA-B57 polypeptide is joined with the IgG polypeptide as part of a single polypeptide chain by a peptide linker, a short sequence of amino acids 5, 10, 15, or 20, or even 50 residues in length. In particular embodiments, the peptide linker is a non-immunogenic sequence rich in serine and glycine residues. In more particular embodiments, the peptide linker has the sequence SEQ ID NO 003.
Natural HLA molecules expressed in the endoplasmic reticulum of human cells associate with peptide epitopes before undergoing transport and display on the cell surface. In particular embodiments of the pharmaceutical composition according to the invention, the HLA polypeptide is not associated with a peptide epitope. In other words, the antigen-binding groove, or antigen-binding cleft formed by the alpha 1 and alpha 2 domains of the HLA polypeptide is not bound to a small, antigenic peptide. Binding of peptide to HLA class molecules is thought to change their conformation, which may affect interactions with binding partners such as LILRB1 and LILRB2. The binding affinity and immunomodulatory effects of an HLA-B57 polypeptide associated with β2m polypeptide according to the invention, not further associated with a peptide epitope are demonstrated, for example, in
In alternative embodiments of the method to produce an HLA fusion protein, or the isolated HLA fusion protein according to the invention, the HLA fusion protein additionally comprises a peptide epitope fragment.
Another aspect of the invention provides a nucleic acid encoding the isolated HLA fusion protein according to any one if the aspects or embodiments of the invention recited above, where the encoded variant HLA-B57 polypeptide portion of the HLA fusion protein is characterized by an E46, and an R97. In particular embodiments, the nucleic acid encoding the HLA fusion protein comprises the sequence SEQ ID NO 016. In particular embodiments the nucleic acid also encodes a secretory signal permitting secretion of the cell to enable efficient purification of the HLA fusion protein. In other particular embodiments, the nucleic acid encoding the isolated fusion protein comprises the sequence designated SEQ ID NO 006, further encoding a secretory signal. In more particular embodiments, it essentially consists of SEQ ID NO 006.
Further aspects of the invention include a nucleic acid expression vector comprising the nucleic acid as specified above, and a promoter sequence operable in a cell. In particular embodiments, the promotor is suitable for expression in a eukaryotic cell. In more particular embodiments, a promotor for a mammalian cell is used.
A further aspect of the invention is an isolated cell comprising the HLA fusion protein, or a nucleic acid encoding the HLA fusion protein according to any previous aspects of the invention. These aspects of the invention encompass DNA or RNA-based delivery systems for HLA fusion proteins, or transfer of cells expressing the HLA fusion protein.
The data presented in the examples shows that a variant HLA fusion protein according to the invention, where the A46, and V97 of the extracellular domains of the HLA-B*57:01 polypeptide have been replaced by an E46, and an R97 by targeted amino acid substitutions, binds to LILRB2, increases the killing capacity of CD8 T cells, and provokes anti-tumor immune responses in vivo. The amino acid substitutions in the variant HLA-B57 polypeptide provided by the invention are advantageous in that they increase the yield of the resulting HLA fusion protein comprising said variant HLA-B57 polypeptide, while maintaining the immunomodulatory effects of the molecule, including T cell killing, macrophage activation and phagocytosis, and in vivo therapeutic efficacy (
One aspect of the invention relates to a pharmaceutical composition comprising at least one of the HLA fusion proteins, or the nucleic acid encoding the HLA fusion protein as specified herein, and at least one pharmaceutically acceptable carrier, diluent or excipient, for use in treating a malignant neoplastic disease. In some embodiments, the pharmaceutical composition comprises at least two pharmaceutically acceptable carriers, such as those described herein.
In particular embodiments, the malignant neoplastic disease in which the pharmaceutical HLA fusion protein formulation is used is a solid cancer. In more particular embodiments, the solid cancer is lung cancer, or metastatic lung cancer. In other particular embodiments, the cancer is a form colorectal cancer, or metastatic colon cancer.
In other particular embodiments, the pharmaceutical HLA fusion protein formulation is used to treat a blood-cell derived cancer. In more particular embodiments, it is used to treat T cell leukemia.
In certain embodiments of the invention, the HLA fusion protein of the present invention is typically formulated into pharmaceutical dosage forms to provide an easily controllable dosage of the drug and to give the patient an elegant and easily handled product.
The dosage regimen for the HLA fusion protein of the present invention will vary depending upon known factors, such as the pharmacodynamic characteristics of the particular agent and its mode and route of administration; the species, age, sex, health, medical condition, and weight of the recipient; the nature and extent of the symptoms; the kind of concurrent treatment; the frequency of treatment; the route of administration, the renal and hepatic function of the patient, and the effect desired. In certain embodiments, the HLA fusion protein of the invention may be administered in a single daily dose, or the total daily dosage may be administered in divided doses of two, three, or four times daily.
Many procedures and methods for preparing pharmaceutical compositions are known in the art, see for example L. Lachman et al. The Theory and Practice of Industrial Pharmacy, 4th Ed, 2013 (ISBN 8123922892).
Another aspect of the invention is pharmaceutical composition comprising a variant HLA fusion protein according to the invention, or a nucleic acid encoding said HLA fusion protein, formulated for administration in combination with a checkpoint inhibitory agent. The inventors have previously found HLA-B57 IgG4 fusion protein compounds to enhance the effect of checkpoint inhibitors, and reason similar molecules based on the variant HLA-B57 heavy chain polypeptide will provide a similar effect when administered in combination, as the molecule shows similar immunomodulatory action in the in vitro assays tested in the examples.
Another aspect of the invention is a checkpoint inhibitor for use in treating a malignant neoplastic disease, formulated for administration in combination with an HLA fusion protein, or a nucleic acid, or a vector encoding the fusion protein according to the invention.
The checkpoint inhibitory agent according to the invention is capable of disrupting the inhibitory signaling cascade that limits immune cell activation, and in particular T cell activation. In certain embodiments, the checkpoint inhibitory agent is an antibody fragment, or an antibody-like molecule capable of binding to one of CTLA-4, PD-1, PD-L1, or PD-L2 with a dissociation constant of 10−7 mol/L or lower (higher affinity).
In the context of the present specification, the term dissociation constant (KD) is used in its meaning known in the art of chemistry and physics; it refers to an equilibrium constant that measures the propensity of a complex composed of [mostly two] different components to dissociate reversibly into its constituent components. The complex can be e.g. an antibody-antigen complex AbAg composed of antibody Ab and antigen Ag. KD is expressed in molar concentration [mol/L] and corresponds to the concentration of [Ab] at which half of the binding sites of [Ag] are occupied, in other words, the concentration of unbound [Ab] equals the concentration of the [AbAg] complex. The dissociation constant can be calculated according to the following formula:
[Ab]: concentration of antibody; [Ag]: concentration of antigen; [AbAg]: concentration of antibody-antigen complex
Particularly, the checkpoint inhibitor agent is a non-agonist CTLA-4 ligand, a non-agonist PD-1 ligand, a non-agonist PD-L1 ligand, or a non-agonist PD-L2 ligand, which does not lead to attenuated T cell activity when binding to CTLA-4, PD-1, PD-L1 or PD-L2, respectively, on the surface on a T-cell. In certain embodiments, non-agonist CTLA-4 ligands used in the present invention are able, when bound to CTLA-4, to sterically block interaction of CTLA-4 with its binding partners CD80 and/or CD86 and the non-agonist PD-1 ligands used in the present invention are able, when bound to PD-1, to sterically block interaction of PD-1 with its binding partners PD-L1 and/or PD-L2.
In certain embodiments, the checkpoint inhibitory agent disrupts inhibitory signaling cascades via a capacity to bind to CTLA-4 with a dissociation constant of, to mark the least affinity as expressed in KD value, 10−7 mol/L, particularly of 10−8 mol/L or even 10−9 mol/L and which inhibits the biological activity of its respective target. A non-agonist PD-1 ligand or a non-agonist PD-L1 (PD-L2) ligand in the sense of the invention refers to a molecule that is capable of binding to PD-1 (PD-L1, PD-L2) with a dissociation constant of at least 10−7 mol/L, particularly 10−8 mol/L or even 10−9 mol/L or lower, and which inhibits the biological activity of its respective target. In particular embodiments, the checkpoint inhibitory agent is an antibody, more particularly an antibody specific for PD-1, which inhibits interactions with PD-L1 to enhance T cell responses.
In particular embodiments of the pharmaceutical composition for combination treatment, the checkpoint inhibitory agent is a checkpoint inhibitor antibody selected from the clinically available antibody drugs ipilimumab (Bristol-Myers Squibb; CAS No. 477202-00-9), tremelimumab (CAS 745013-59-6), nivolumab (Bristol-Myers Squibb; CAS No 946414-94-4), pembrolizumab (Merck Inc.; CAS No. 1374853-91-4), pidilizumab (CAS No. 1036730-42-3), atezolizumab (Roche AG; CAS No. 1380723-44-3), avelumab (Merck KGaA; CAS No. 1537032-82-8), durvalumab (Astra Zeneca, CAS No. 1428935-60-7), and cemiplimab (Sanofi Aventis; CAS No. 1801342-60-8).
In certain embodiments, the checkpoint inhibitor agent is characterized by specific binding for the 4-1BB protein. In particular embodiments, the checkpoint inhibitor is a clinically available antibody drug selected from utomilumab (CAS No. 1417318-27-4), or urelumab (CAS No. 934823-49-1).
Previous disclosures by the inventors in WO 2017153438 A1 show that soluble HLA heavy chain peptides reduce tumor burdens in multiple tumor models alone, or when paired with checkpoint inhibitory agents characterized by specific binding to PD-1, PD-L1, and 4-1 BB.
An antibody fragment may be a Fab domain or a variable fragment (Fv) domain of an antibody, or a single-chain antibody fragment (a fusion protein consisting of the variable regions of light and heavy chains of an antibody connected by a peptide linker). The inhibitor may also be a single domain antibody, consisting of an isolated variable domain from a heavy or light chain. Additionally, an antibody may also be a heavy-chain antibody consisting of only heavy chains such as antibodies found in camelids. An antibody-like molecule may be a repeat protein, such as a designed ankyrin repeat protein (Molecular Partners, Zurich).
In certain embodiments, the combination therapy comprises two distinct dosage forms, for example, said HLA fusion protein is provided as a dosage form for intra-tumoral delivery, or local delivery in the vicinity of the tumor, for example, by subcutaneous injection, or intra-tumoral injection into a solid tumor, and said checkpoint inhibitor agent is provided as a dosage form for systemic delivery, particularly by intravenous injection. However, said checkpoint inhibitor agent and said HLA fusion protein may also be delivered in two similar dosage forms.
Administration in combination, encompasses both simultaneous administration of the checkpoint inhibitor agent and the HLA fusion protein together, or in separate formulations, or administration of one substance immediately prior to, for example, in the week prior to, or immediately subsequent to, for example, in the week subsequent to, administration of a second substance. In some embodiments, administration of the two agents in combination refers to administration of one agent prior to, particularly in the month prior to the second agent. In other embodiments, one second is administered subsequent to, particularly in the weeks, or month subsequent to, the first agent. In other particular embodiments, the checkpoint inhibitor and the HLA fusion protein are administered in overlapping administration regimes.
The invention further encompasses a pharmaceutical composition comprising an HLA fusion protein according to the invention, and a checkpoint inhibitory agent.
The invention further encompasses a pharmaceutical composition comprising an HLA fusion protein for use in treating a patient having recently being administered, or scheduled to receive a checkpoint inhibitory agent.
The invention further encompasses a pharmaceutical composition comprising a checkpoint inhibitory agent for use in treating a patient having recently being administered, or scheduled to receive an HLA fusion protein.
In certain aspects and embodiments, the variant HLA-B57 polypeptide according to the invention is provided for use in treating various forms of cancer.
In particular embodiments of the pharmaceutical composition comprising an HLA fusion protein, it is provided for use to treat a type of liquid, or blood cancer. The inventors consider the characteristic T cell-exhaustion, and accessibility of circulating blood cancer cells to the T cell, and macrophage activation induced by pharmaceutical compositions according to the invention mean this combination is likely to be efficacious. In particular such embodiments, the pharmaceutical composition is provided for use in a patient diagnosed with T cell leukemia, as modelled herein with Jurkat cells. In other particular embodiments, the pharmaceutical composition is provided for use in a patient diagnosed with multiple myeloma, as modelled by the THP-1 AML cell line.
In other particular embodiments, the pharmaceutical composition comprising an HLA fusion protein is provided for use in a patient diagnosed with a solid cancer, or a metastasis of a solid cancer. In some particular embodiments, the pharmaceutical composition is for use in a patient diagnosed with lung cancer. In particular embodiments, the lung cancer is a form of non-small cell lung cancer. In other particular embodiments, the lung cancer is a form of small cell lung cancer, or carcinoma. In other particular embodiments, the pharmaceutical composition comprising and HLA fusion protein, or the immune checkpoint inhibitor is for use in a patient diagnosed with colon cancer. In particular embodiments, metastatic colon cancer.
In some embodiments of aspects of the invention relating to administration of a pharmaceutical composition according to the invention in addition to a checkpoint inhibition agent, it is provided for use in a form of malignant disease, or cancer, in which checkpoint inhibition therapy is approved for monotherapy, or combination therapy. In particular embodiments, the combination treatment is for use in a patient with colon cancer, particularly metastatic colon cancer. On other particular embodiments, the cancer is melanoma. In further particular embodiments, the cancer is pancreatic cancer. In still further particular embodiments, the cancer is breast cancer.
The invention further encompasses, as an additional alternative aspect, the use of an HLA fusion protein, or of a pharmaceutical composition comprising an HLA fusion protein as specified in detail above, for use in a method of manufacture of a medicament for the treatment, or prevention of recurrence, of a form of cancer. Another alternative aspect, is a method of treating a patient diagnosed with a form of malignant neoplastic disease by administering an effective amount of a pharmaceutical composition comprising an HLA fusion protein as specified in detail above. The invention further encompasses co-administration of a pharmaceutical composition comprising an HLA fusion protein according to the invention, and a pharmaceutical composition comprising a checkpoint inhibitory agent to a cancer patient. The invention further encompasses administration of a pharmaceutical composition comprising an HLA fusion protein according to the invention, to a patient who has already been administered a pharmaceutical composition comprising a checkpoint inhibitory agent.
Dosage forms may be for parenteral administration such as subcutaneous, intravenous, intrahepatic or intramuscular injection forms. Optionally, a pharmaceutically acceptable carrier and/or excipient may be present.
The invention further encompasses the following items:
Wherever alternatives for single separable features are laid out herein as “embodiments”, it is to be understood that such alternatives may be combined freely to form discrete embodiments of the invention disclosed herein.
The invention is further illustrated by the following examples and figures, from which further embodiments and advantages can be drawn. These examples are meant to illustrate the invention but not to limit its scope.
CDNAs encoding HLA-B57-Fc (SEQ ID NO 012) & HLA-B57(A46E/V97R)-Fc (SEQ ID NO 006) preceded by secretory leader signals were cloned separately into expression vectors (Probiogen). The vector constructs expressing HLA-B57-Fc & HLA-B57(A46E/V97R)-Fc were co-transfected with a plasmid comprising a nucleic acid (SEQ ID NO 013) encoding the β2m protein (SEQ ID NO 014) by microporation (MP) using the NEON Transfection Kit (Life Technologies #MPK10096). Using the same process, nucleic acid expression vectors encoding alternative immunogenic HLA class I heavy chains IgG4 fusion proteins were created comprising the extracellular domain of HLA-A30:01 (SEQ ID NO 021), HLA-B58:01 (SEQ ID NO 022), or HLA-Cw08:02 (SEQ ID NO 023), or variant extracellular domains characterized by single, or double amino acid substitutions at position 46 and 97 of the HLA heavy chain polypeptide. Modified constructs were created introducing to measure the impact amino acid substitutions adding, or removing an E46 amino acid residue, or a R97 residue into the HLA heavy chain extracellular domain portion of each HLA-Fc fusion protein as follows: HLA-A30E46A (SEQ ID NO 024), HLA-A30I97R (SEQ ID NO 025), HLA-A30E46A/I97R (SEQ ID NO 026), HLA-B57A46E (SEQ ID NO 027), HLA-B57V97R (SEQ ID NO 028), HLA-B57A46E/V97R (SEQ ID NO 015), HLA-B58E46A (SEQ ID NO 029), HLA-B58R97V (SEQ ID NO 030), HLA-B58E46A/R97V (SEQ ID NO 031), HLA-C08E46A (SEQ ID NO 032), HLA-C08R97V (SEQ ID NO 033), HLA-C08E46A/R97V (SEQ ID NO 034).
CHO-DG44 starter cells were transfected at different ratios of HLA-Fc to β2m plasmid (4:1, 2:1, 1:1, 1:2). Selected clone pools were grown in standardized shaker flasks and with a defined cell seeding density of 4E5 vc/mL in 125 mL of PBG-CD-C4 supplemented medium including puromycin and methotrexate. Following adjusted selection pression with antibiotics, individual clone pools were selected for analysis. Measurement of viabilities and viable cell densities were performed using the Vi-CELL XR System, and Trypan blue cell exclusion method. Titer quantifications were measured at different time points (days) using an Octect RED machine (ForteBio, a Pall Division) with Protein A biosensors.
Purification of HLA-B57.β2m and HLA-B57(A46E/V97R).β2m and/β2m Removal Procedure
Filtered supernatants containing the secreted HLA-B57.β2m and HLA-B57(A46E/V97R).β2m were used for affinity column purification. Purification of proteins and removal of β2m under acidic conditions was performed as a two-step purification protocol. As a first step, Protein G Sepharose [(4 Fast Flow) Sigma, #GE-17-0618-01)] beads were used to capture HLA-B57+β2m and HLA-B57(A46E/V97R)+β2m from supernatants. After an overnight incubation at 4 degrees on a rocker, the recovered beads were washed in PBS, and subsequently proteins were eluted using standard IgG-Elution Buffer (pH 2.8) (Pierce™ IgG Elution Buffer, Thermo Fischer #21004). A second step of size exclusion chromatography-based purification was performed to separate HLA-B57 & HLA-B57(A46E/V97R) from R2m under acidic conditions. A Superdex 10/300 gel filtration column, pre-equilibrated in Sodium Citrate (100 mM, pH 3.0) was used for the separation. An injection of 0.5 ml of the protein at 2.0 mg/ml concentration was applied, and the desired non-β2m-associated HLA-B57 (SEQ ID NO 018) & HLA-B57(A46E/V97R) (SEQ ID NO 015) protein peaks eluted at 12.7 ml and the peak for β2m eluted at 22.0 ml. These results demonstrate that the separation of β2m and purification of non-β2m-associated HLA-B57 and HLA-B57(A46E/V97R) is feasible under acidic conditions.
The quantification of the affinity of interaction of LILRB2 with non-β2m-associated HLA-B57, and HLA-B57(A46E/V97R) was conducted using the enzyme-linked immunosorbent assay (ELISA) method. Flat bottom Pierce™ Streptavidin coated high binding capacity 96 well plates (Pierce #15500) were used and 50 μl of c-terminally biotinylated antigen molecules (LILRB2, BPS Bioscience #100335) was immobilized at a final concentration of 5 μg/ml in PBS buffer. PBS and IgG isotype were used as negative controls. A serial dilution of non-β2m-associated HLA-B57, or HLA-B57(A46E/V97R) (eight concentration points: 10, 2.5, 1, 0.25, 0.1, 0.025, 0.01, 0.0025 μg/ml) was applied (50 μl) in duplicate. An APC conjugated goat anti-human IgG antibody (Jackson Immuno Research #109-135-098) with 1:100 dilution in TBS (50 μl) was used for detection. Finally, 50 μl TBS in each well was added and a fluorescence scan was performed with APC excitation and emission wavelengths of 650 nm & 660 nm, respectively. Using Graphpad Prism v9.1.2, a three-parameter based log (agonist) vs. response model was used to determine the EC50 of the interaction.
Flow cytometry was performed on an LSR Fortessa Analyzer (BD Biosciences). T cell surface markers CD3, CD4 and CD8 were assessed by antibody staining (BioLegend) at a dilution of 1:100. HLA-DR expression on macrophages was assessed by antibody staining (BioLegend) at a dilution of 1:100. The macrophage polarization panel was performed using CD80, CD86, CD68, CD163, CD206 and CD209 antibodies (Biolegend) at a dilution of 1:100. All stains were performed on ice for 20 min, then were washed and resuspended according to standard practice.
For the co-culture assay, human T cells were isolated from peripheral blood mononuclear cells (PBMCs) from healthy donors, stimulated with CD3/CD28-activator (ThermoFisher #11131D) and cultured in the presence of 50 U/ml rhIL-2 for 48 hours. T cells were then washed from the CD3/CD28-activator and subsequently mixed with the indicated human leukemia cells in a U-bottom 96-well plate in duplicate wells. Compounds were added at indicated concentrations to each well. Leukemia cells were stained prior to co-culture with CellTrace™ violet cell proliferation marker (ThermoFisher #C34557) according to manufacturer's instruction. The number of the plated cells, the E:T ratio and the duration of the co-cultures was tested for different leukemia cell lines and is indicated in the associated figure. Co-cultures were photographed using an inverted microscope, and T cells were stained with CD3, CD4 and CD8 antibodies and analyzed by LSR Fortessa Analyzer. Live cancer cells were positive for violet cell proliferation marker and negative for sytox red dead cell stain (ThermoFisher #S34859). Absolute count of both T cells and violet stain-positive cancer cells was measured using Bright count beads (ThermoFisher #C36590).
Primary human donor-derived monocytes were isolated from PBMCs from a healthy donor and differentiated into macrophages by 5-7 days of culture in ImmunoCult medium (StemCell Technologies #10961)+50 ug/ml rhMCSF (StemCell #78057.1). On day 1 post plating compounds were added to wells at a concentration of 20 μg/ml. On day 5-7 post plating, compounds were once again added to the macrophages and two downstream experiments were performed: 1) polarization of macrophages: For polarization studies cultured macrophages were analyzed by flowcytometry for expression of CD80, CD86, CD68, CD163, CD206 and CD209 (Biolegend) one day after the second treatment with compounds. 2) Phagocytosis: Target cells were plated on macrophages at a ratio of 0.5×106 to 1×106 macrophages in 12 well plates and analyzed for phagocytosis 16 hours post plating. Target cells were stained with CellTrace™ violet cell proliferation marker prior to co-culture and thus could be differentiated from macrophages which were stained with HLA-DR by flow cytometry. Phagocytosis was evaluated as the percentage of violet stain-positive target cells from HLA-DR positive macrophages, as analyzed using FlowJo v.10.6.1 (Tree Star). Each phagocytosis reaction was performed in technical duplicates. All biological replicates indicate independent human macrophage donors.
C38 tumor fragments were injected subcutaneously into the right flanks of syngeneic female C57BL/6 mice. Once the tumor reached ±50 mm3 in colon animals were distributed according to their individual tumor volume size and divided into groups displaying no statistical differences between them. Tumor diameters were measured using a caliper, and volume was calculated according to the formula, D/2×d2 where D and d are the longest and shortest diameter of the tumor in mm, respectively. The Experimental design of injection time points of cells and injection of substances was established as follows: isotype IgG4 (10 mg/Kg) biwk×3; non-β2m-associated HLA-B57(A46E/V97R) (10 mg/Kg) biwk×3; Biwk=bi-weekly
To optimize the expression profile of HLA-heavy chain non-β2m-associated Ig Fc fusion proteins for medical use, the protein sequences of a range of HLA heavy chain sequences were aligned to identify residues potentially associated with increased yield in a transient transfection system using Chinese hamster ovary cells (CHO). This comparison showed that specific glutamic acid (E) and arginine (R) amino acid residues present in diverse HLA molecules correlated with increase expression as measured by the concentration of recombinant protein measured in supernatant, suggesting these amino acid substitution changes may confer a variant HLA-B57(A46E/V97R) with the superior expression levels of related HLA proteins when expressed in mammalian cells (
The HLA-B57 haplotype is characterized by genetic linkage to immune phenotypes, and binds to innate receptors including the immunoglobulin-like receptor subfamily B member 2 (LILRB2), making the HLA-B57 protein sequence a particularly desirable HLA-heavy chain component for use in an HLA fusion protein. Substitution mutations were introduced into the amino acid sequence of an HLA-IgG4 Fc fusion protein based on the HLA-B57 alpha 1, 2 and 3 domains of the naturally occurring HLA-B57:01 protein (SEQ ID NO 001), exchanging an A at position 46 with an E, and a V at position 97 with an R (
To test whether substitute amino acid residues affected the expression, or yield of recombinant HLA heavy chain IgG4 fusion proteins, CHO cell clones expressing the HLA-B57(A46E/V97R)-Fc and HLA-B57-Fc construct were isolated and sub cloned, and the concentration of the resulting complexes comprising β2m together with either the wildtype (HLA-B57.β2m) or mutant HLA-B57 (HLA-B57(A46E/V97R)).β2m) fusion proteins was assessed by Protein A biosensors (Octet Red96 system, Sartorius). HLA-B57(A46E/V97R)).β2m-producing clones showed increased cell and produced a significantly higher titer of HLA fusion protein in supernatant compared to HLA-B57:β2m-expressing control cells (
The activity of HLA-B57(A46E/V97R) was compared to the HLA-B57(A46E/V97R) parent structure in several assays to confirm the biological activity of HLA domain of an HLA IgG4 Fc fusion protein was not compromised by the addition of the two amino acid changes. ELISA analysis of non-β2m-associated compounds based on the non-variant or variant HLA-B57 heavy chain sequence demonstrated that the two amino acid substitutions did not reduce binding to LILRB2, and indeed the variant HLA fusion protein showed an improved, lower EC50 value of LILRB2 binding saturation (
To confirm the importance of 46E and 97R residues for optimal recombinant expression of various HLA class I heavy chains associated with differing immune phenotypes in the human population, the inventors measured the impact of these amino acid substitutions on additional IgG Fc fusion protein constructs comprising additional HLA class I heavy chain polypeptide sequences associated with different immunogenic effects in the human population, HLA-A30, HLA-B58, and HLA-C08 (
These results confirmed that in all molecules tested, HLA heavy chains characterized by both amino acid 46E and 97R residues were associated with optimal recombinant protein yields, and that introducing both an A46E and a V97R substitution into the HLA-B57 heavy chain sequence achieved the highest yield among all constructs. Conversely, the introduction of both 46A and 97V present in wildtype HLA-B57 into HLA-A30, HLA-B58, or HLA-C08 significantly reduced productivity yields, confirming that amino acids 46E and 97R are key for stabilization and production of optimal titers of HLA heavy chains, including HLA-Fc molecules.
AAAMNFGLRLIFLVLTLKGVQCGSHSMRYFYTAMSRPGRGEPRFIAVGYVDDTQFVRFDSDAAS
FLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCL
VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEA
LHNHYTQKSLSLSLG
FPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVL
TVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVK
GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALH
NHYTQKSLSLSLG
VFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV
SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTC
LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHE
ALHNHYTQKSLSLSLG
| Number | Date | Country | Kind |
|---|---|---|---|
| 21190004.8 | Aug 2021 | EP | regional |
| 21190005.5 | Aug 2021 | EP | regional |
| 21207324.1 | Nov 2021 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2022/072131 | 8/5/2022 | WO |
