The present invention relates to methods of producing human IgG antibodies, particularly IgG1 antibodies, including fragment thereof, with enhanced effector functions.
Formation of an antibody-antigen complex and recognition by specialized immune cells triggers a wide range of immune system responses. The most common antibody isotype is IgG, composed of two identical heavy chains that are disulfide linked to two identical light chains. Antigen recognition occurs in the complementarity determining region formed at the terminal end of the associated heavy and light chains. At the other antibody terminus, interactions initiated through the binding of the antibody Fc domain to Fc receptors, leads to Fc effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC), cell-mediated complement activation (CDC), and phagocytosis (opsonization).
Fc receptors are cell-surface glycoproteins found on particular immune cells that can bind the terminal Fc portion of an antibody. These Fc receptors are defined by their distribution, immunoglobulin subtypes specificity and the effector response initiated. For example, FcγR receptors found on macrophages, peripheral blood mononuclear cells (PBMCs), and natural killer cells (NK) are more specific for IgG type molecules. NK cell FcRγIIIa receptor binding of the antibody bound target then mediates ADCC target cytolysis. Activation of the complement cascade on the other hand, is initiated by binding of serum complement protein C1q to the Fc portion of an antibody-antigen complex. Though not a cell surface molecule, C1q can still be considered an Fc receptor as C1q can direct either CDC or phagocytosis by recruiting deposition of the C3 complement component, followed by recognition by C3 receptors on various phagocytic cells.
Each human IgG heavy chain has an antigen recognizing variable domain (V) and 3 homologous constant-region domains; CH1, CH2 and CH3 where the CH2 and CH3 comprise the Fc region. Mutagenesis studies have shown that it is the CH2 and CH3 domains that most important to these Fc receptor mediated responses. Thus, by identifying the key Fc amino acid residues mediating Fc receptor interactions, antibody Fc engineering could potentially provide new capabilities and improvements to selectively increase Fc-effector functions, alter FcR targeting for more efficient radionuclide or cytotoxic drug targeting and/or optimize therapeutic half life modalities requiring chronic dosing regimens.
It would thus be desirable to provide a systematic mutagenesis and screening method by which beneficial mutations throughout the entire Fc region for chosen Fc effector properties can be rapidly and efficiently identified. To facilitate the screening method, it would be further desirable to provide a method in which Fc mutations are expressed as a mammalian Fc variant library on the surface of mammalian cells, such that the Fc variants can be directly screened by in vitro ADCC and/or CDC assay readouts.
The invention includes, in one aspect, a method of generating human IgG1 antibodies with enhanced effector function. In carrying out the method, there is constructed an IgG1 Fc look-through mutagenesis (LTM) coding library. The library may be a regional LTM library encoding, for at least one of the two IgG1 Fc regions identified by SEQ ID NOS: 1 and 2, representing the CH2 and CH3 regions of the antibody's Fc fragment, respectively, and for each of a plurality of amino acids, individual amino acid substitutions at multiple amino acid positions within one of the two IgG1 Fc regions. Alternatively, the library may be a sub-region LTM library encoding, for each of the four regions identified by SEQ ID NOS: 14-17 contained within the IgG1 Fc CH2 region identified by SEQ ID NO:1, and for each of a plurality of selected amino acids, individual substitutions at multiple amino acid positions within each region.
The IgG1 Fc fragments encoded by the LTM library are expressed in a selectable expression system, and those expressed IgG1 Fc fragments that are characterized by an enhanced effector function are selected. The enhanced effector function is related to (i) a shift in binding affinity constant (KD), with respect to a selected IgG1 Fc binding protein, relative to native IgG1 Fc; or (ii) a shift in the binding off-rate constant (Koff); with respect to a selected IgG1 Fc binding protein, relative to native IgG1 Fc, and may be based on either a direct KD or Koff measurement or an indirect measure of binding, such as antibody-dependent cell-mediated cytotoxicity (ADCC), cell-mediated complement activation (CDC), and phagocytosis (opsonization).
The expressed Fc fragments encoded by the library may be expressed in a selectable expression system composed of viral particles, prokaryotic cells, and eukaryotic cells, where the expressed Fc particles are attached to the surface of the expression-system particles and accessible thereon to binding by the Fc binding protein. One exemplary expression system includes a mammalian cell, such as a BaF3, FDCP1, CHO, and NSO cell, that is (i) capable of producing clinical-grade monoclonal antibodies, (ii) nonadherent in culture, and (iii) readily transduced with a retrovirus.
The expression system may include a mammalian cell that expresses the Fc fragments on its surface, allowing a direct measure of Fc effector function, such as antibody-dependent cell-mediated cytotoxicity (ADCC), cell-mediated complement activation (CDC), and phagocytosis (opsonization). This direct method includes the steps of (i) adding expression cells corresponding to a single clonal variant of the LTM library to each of a plurality of assay wells, (ii) adding to each well, reagents that include an Fc binding protein and which are effective to interact with the surface-attached Fc fragment, and depending on the level of binding thereto, to lyse the cells, (iii) assaying the contents of the wells for the presence of cell lysis products, and (iv) selecting those IgG1 Fc fragments which are expressed on cells showing the greatest level of cell lysis.
For measuring ADCC directly, the reagents added in step (ii) may be peripheral blood mononuclear cells capable of lysing cells expressing the Fc fragment on their surface by antibody-dependent cellular cytotoxicity. The method may further include, prior to step (i), enriching such cells for those expressing Fc fragments having an elevated binding affinity constant or reduced binding off-rate constant, with respect to Fc-binding proteins FcγRI or FcγRIIIa.
For measuring CDC directly, the reagents added in step (ii) are human C1q complex and human serum, capable of lysing cells by complement-mediated cell death. The method may further include, prior to step (i), enriching such cells for those expressing Fc fragments having an elevated binding affinity constant or reduced binding off-rate constant, with respect to Fc-binding protein C1q.
In both cases of direct measuring of effector function, the method may further include enriching the cells for those expressing Fc fragments having one of: (i) an elevated binding affinity constant or reduced binding off-rate constant, with respect to Fc-binding protein C1q, FcγRI, FcγRIIa, and FcγRIIIa, (ii) a reduced binding affinity constant or elevated binding off-rate constant with respect to Fc-binding proteins FcγRIIb, FcγRIIIb; and (iii) an elevated or reduced binding affinity constant or a reduced or elevated binding off-rate constant, respectively, with respect to Fc-binding protein FcRN and protein A.
For generating expressed Fc fragments having an elevated binding affinity constant, with respect to Fc-binding protein selected from the group consisting of C1q, FcγRI, FcγRIIa, FcγRIIIa, FcRN and protein, relative to the binding affinity constant for native IgG1 Fc fragment, the selecting step may include (i) forming a mixture of expression particles with displayed Fc fragments and an Fc binding protein, (ii) allowing the Fc binding protein to bind with the displayed Fc fragments in the mixture, to form an Fc-binding complex, and (iii) isolating the Fc-binding complexes from the mixture, wherein particles expressing Fc fragments having the highest binding affinity constants for the binding protein are isolated.
For generating Fc fragments having an elevated equilibrium binding affinity constant, with respect to Fc-binding protein selected from the group consisting of C1q, FcγRI, FcγRIIa, FcγRIIIa, FcRN and protein A, relative to the binding affinity constant for native IgG1 Fc fragment, the selecting may step include (i) forming a mixture of expression particles with displayed Fc fragments and a limiting amount of fluorescent-labeled Fc binding protein in soluble form, such that those particles expressing Fc fragments with a higher binding affinity constant will be more strongly labeled, (ii) after the binding in the mixtures reaches equilibrium, sorting the particles on the basis of amount of bound fluorescent label, and (iii), selecting those particles having the highest levels of bound fluorescence.
For generating Fc fragments having a reduced binding off-rate constant, with respect to Fc-binding protein selected from the group consisting of FcγRIIb, FcγRIIIb, FcRN and protein A, relative to the binding affinity constant for native IgG1 Fc fragment, the selecting step may include (i) forming a mixture of expression particles with displayed Fc fragments and a limiting amount of fluorescent-labeled Fc binding protein in soluble form, such that those particles expressing Fc fragments with a lower binding affinity constant will be less strongly labeled, (ii) after the binding in the mixtures reaches equilibrium, sort the particles on the basis of amount of bound fluorescent label, and (iii), selecting those particles having the lowest levels of bound fluorescence.
For generating Fc fragments having a reduced binding off-rate affinity constant, with respect to Fc-binding protein selected from the group consisting of C1q, FcγRI, FcγRIIa, FcγRIIIa, FcRN and protein A, relative to the binding affinity constant for native IgG1 Fc fragment, the selecting step may include (i) forming a mixture of expression particles with displayed Fc fragments and a saturating amount of fluorescent-labeled Fc binding protein in soluble form, (ii) at a selected time after step (i), adding a saturating amount of an unlabeled Fc binding protein, (iii) at a selected time after step (ii) and prior to binding equilibrium, sort the particles on the basis of amount of bound fluorescent label, and (iv), selecting those particles having the highest levels of bound fluorescence.
For generating Fc fragments having an increased binding off-rate affinity constant, with respect to Fc-binding protein selected from the group consisting of FcγRIIb, FcγRIIIb, FcRN and protein A, relative to the binding affinity constant for native IgG1 Fc fragment, the method may include (i) forming a mixture of expression particles with displayed Fc fragments and a saturating amount of fluorescent-labeled Fc binding protein in soluble form, (ii) at a selected time after step (i), adding a saturating amount of an unlabeled Fc binding protein, (iii) at a selected time after step (cii) and prior to binding equilibrium, sort the particles on the basis of amount of bound fluorescent label, and (iv), selecting those particles having the lowest levels of bound fluorescence.
For generating Fc fragments having the ability, when incorporated into an IgG1 antibody, to enhance antibody-dependent cellular-toxicity, the method may further include, after identifying IgG1 Fc fragments characterized by an elevated binding affinity constant or reduced binding off-rate constant for FcγRIIIA, the selecting step may further include selecting the identified fragments for binding affinity for the FcγRIIB receptor that exhibits reduced binding affinity constant or elevated binding off-rate constant for the FcγRIIB receptor.
For generating Fc fragments having the ability, when incorporated into an IgG1 antibody, to enhance complement-dependent cytotoxicity (CDC), wherein step (c) further includes, after identifying IgG1 Fc fragments characterized by an elevated binding affinity constant or reduced binding off-rate constant for C1q complex, the selecting step may further include selecting the identified fragments for binding affinity for the FcγRIIB receptor that exhibits reduced binding affinity constant or elevated binding off-rate constant for the FcγRIIB receptor. For generating Fc fragments having the ability, when incorporated into an exogenous therapeutic IgG1 antibody, to enhance the therapeutic response to the antibody in human patients having a position position-158 receptor polymorphism in the FcγRIIIA receptor, the selecting step may include selecting those expressed IgG1 Fc fragments that are characterized by a binding affinity for the FcγRIIIA F158 receptor polymorphism that is at least as great as that for a FcγRIIIA V158 receptor polymorphism.
For generating Fc fragments having the ability, when incorporated into an exogenous therapeutic IgG1 antibody, to enhance the therapeutic response to the antibody in human patients having a position-134 receptor polymorphism in the FcγRIIA receptor, the selecting step may include selecting those expressed IgG1 Fc fragments that are characterized by a binding affinity for the FcγRIIA R131 receptor polymorphism that is at least as great as that for a FcγRIIA H131 receptor polymorphism.
The method may further include, after the initial selecting step, the steps of constructing a walk-through mutagenesis (WTM) library encoding, for at least one of the Fc coding regions at which amino acid substitutions are made in the LTM library, the same amino acid substitution at multiple amino acid positions within that region, where the substituted amino acid corresponds to an amino acid variation found in at least one amino acid position of an Fc fragment initially selected; expressing the IgG1 Fc fragments encoded by the WTM library in a selectable expression system; and selecting those IgG1 Fc fragments so expressed that are characterized by a desired shift in binding affinity constant or binding off-rate constant with respect to a selected IgG1 Fc binding protein, compared with the same constant measured for a native Fc fragment.
The IgG1 Fc fragments generated in the method may be characterized by an increased binding affinity constant or reduced binding off-rate constant for a human IgG1 Fc-binding protein, where the shift in constant relative to the same constant measured for a native Fc fragment is greater than a factor of 1.5
The IgG1 Fc fragments generated in the method may be characterized by an decreased binding affinity constant or increased binding off-rate constant for a human IgG1 Fc-binding protein, where the shift in constant relative to the same constant measured for a native Fc fragment is greater than a factor of 1.5
These and other objects and features of the invention will become more fully apparent when the following detailed description of the invention is read in conjunction with the accompanying drawings.
The terms below have the following definitions herein unless indicated otherwise.
The numbering of the residues in an IgG Fc fragment and the heavy chain containing the fragment is that of the EU index as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991), expressly incorporated herein by reference. The “EU index as in Kabat” refers to the residue numbering of the human IgG1 EU antibody.
The term “Fc region” or “Fc fragment” is used to define a C-terminal region of an IgG heavy chain as shown in
The Fc region of an IgG comprises two constant domains, CH2 and CH3, as shown in
“Hinge region” is generally defined as stretching from Glu216 to Pro230 of human IgG1 (Burton, Molec. Immunol. 22:161-206 (1985)) Hinge regions of other IgG isotypes may be aligned with the IgG1 sequence by placing the first and last cysteine residues forming inter-heavy chain S—S bonds in the same positions.
“C1q” is a polypeptide that includes a binding site for the Fc region of an immunoglobulin. C1q together with two serine proteases, C1r and C1s, forms the complex C1, the first component of the complement dependent cytotoxicity (CDC) pathway. Human C1q can be purchased commercially from, e.g. Quidel, San Diego, Calif.
The term “Fc receptor” or “FcR” is used to describe a receptor that binds to the Fc region of an antibody. The preferred FcR is one, which binds an IgG antibody (a γ receptor) and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced forms of these receptors. FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs are encompassed by the term “FcR” herein. The term also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)). The term also include other polypeptides known to binding specifically to the Fc region of an IgG antibody, such as the C1q peptide complex and protein A.
The term “binding domain” refers to the region of a polypeptide that binds to another molecule. In the case of an FcR, the binding domain can comprise a portion of a polypeptide chain thereof (e.g. the α chain thereof) which is responsible for binding an Fc region. One useful binding domain is the extracellular domain of an FcR α chain.
The term “antibody” is used in the broadest sense and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bi-specific antibodies), and antibody fragments so long as they exhibit the desired biological activity.
The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts.
The term “Koff”, as used herein, is intended to refer to the off rate constant for dissociation of an antibody from the antibody/antigen complex, as determined from a kinetic selection set up. The units of a Koff rate constant is sec−1, indicating the rate of dissociation of a binding complex. A higher-valued Koff constant means a higher rate of dissociation and therefore a lower affinity between the two binding species. That is, the affinity between two binding species can be increased by reducing its Koff, and/or increasing its Kon.
The term “KD”, as used herein, refers to the dissociation constant of a particular antibody-antigen interaction, and describes the concentration of antigen (expressed in M) required to occupy one half of all of the antibody-binding sites present in a solution of antibody molecules at equilibrium, and is equal to Koff/Kon, the on and off rate constants for the antibody. The association constant KA of the antibody is 1/KD. The measurement of KD presupposes that all binding agents are in solution. In the case where the antibody is tethered to a cell wall, e.g., in a mammalian-cell expression system, the corresponding equilibrium rate constant is expressed as EC50, which gives a good approximation of KD. A lower the value of KD, the higher the binding constant, i.e., a KD of 10−8 M is greater affinity than 10−7 M.
The three-letter and one-letter amino acid abbreviations and the single-letter nucleotide base abbreviations used herein are according to established convention.
This section describes Fc-LTM libraries employed in the method of the invention. As will be discussed more fully in Section IV below, the purpose of the libraries is to generate selected amino-acid substitution mutations in each or substantially each amino-acid position in one or more selected regions of the Fc fragment, to generate libraries of Fc fragments that can be screened for Fc fragments having enhanced effector function.
The Fc portion or fragment of an IgG antibody 20 are shown in
Two important effector functions for which enhanced Fc function will be screened are cell-mediated cytotoxicity (CDC), and antibody-dependent cellular cytotoxicity ADCC), illustrated in
The LTM libraries and screening methods detailed below are applied specifically to generating enhanced Fc characteristics in IgG1 type antibodies. However, it ill be appreciated that the methods can be applied as well to IgG2, IgG3, and IgG4 subtypes of IgG antibodies, and Section B below discusses various types of enhanced effector function that may be desired with each IgG subtype.
The purpose of look-through mutagenesis (LTM) is to introduce a selected substitution at each of a multiplicity of target mutation positions in a region of a polypeptide. Unlike combinatorial methods or walk-through mutagenesis (WTM), which allow for residue substitutions at each and every position in a single polypeptide (see below), LTM confines substitutions to a single selected position, i.e., a single substitution within a defined region or subregion.
The present invention contemplates two general types of Fc libraries constructed for LTM analysis, both of which are referred to below as an Fc-LTM library. The first library is termed an “unbiased” CH2×CH3 library where each library coding sequence includes an amino acid substitution at one selected residue positions in the CH2 region, and a single amino acid at one selected residue position in the CH3 region, where the library preferably includes, at each or substantially each position in both regions, substitutions for each of a subset of chosen LTM amino acids, which collectively represent the major amino acid classes. That is, rather than examine the effect of all 20 natural L-amino acids; it is more efficient to employ a subset of these that represent the chemical diversity of the entire group. One representative subset of L-amino acids that meets this criterion includes the nine amino acids alanine, aspartate, lysine, leucine, proline, glutamine, serine, tyrosine, and histidine. These amino acids display adequate chemical diversity in size, charge, hydrophobicity, and hydrogen bonding ability to provide meaningful initial information on the chemical functionality needed to improve antibody properties.
As seen in
An alternative scheme for preparing an unbiased library containing a single mutation of one of, e.g., nine amino acids, at one position in each of the CH2 and CH3 domains is illustrated in
The second general type of Fc LTM library represents mutations at positions in one or more of four separate IgG1 Fc-FcγRIIIa “contact” points as identified from the IgG1 Fc-FcγRIIIa co-crystal structure (
After LTM Fc variants are screened and selected using functional assays, the rescue of those clones then allows for identification of that DNA coding sequences, as will be detailed below. In the combinatorial beneficial mutation approach, coding sequences are subsequently generated which represent combinations of the beneficial LTM mutations identified and combines them together into a single library. These combinations may be combinations of different beneficial mutations within a single sub-region or between two or more sub-region within the Fc. Therefore, synergistic effects of multiple mutations can be explored in this process.
The combinatorial approach resembles the Walk Through Mutagenesis method (U.S. Pat. Nos. 5,798,208, 5,830,650, 6,649,340B1 and US20030194807) except that the selected codon substitutions within the Fc sub regions are the different beneficial amino-acid substitutions identified by LTM. As shown in
This section describes methods for generating and expressing Fc-LTM library Fc fragments in accordance with the invention. The design of oligonucleotide LTM and CBM libraries is preferably carried out using software coupled with automated custom-built DNA synthesizers. Implementation of the LTM and CBM strategies involves the following steps. After selection of target amino acids to be incorporated into the selected Fc region(s), the software determines the codon sequence needed to introduce the targeted amino acids at the selected positions. Optimal codon usage is selected for expression in the selected display and screening host, e.g., the mammalian expression system. The software also eliminates any duplication of the wild-type sequence that may be generated by this design process. It then analyzes for potential stop codons, hairpins, loops and other problematic sequences that are then fixed. The software determines the ratios of bases added to each step in the synthesis (for CBM) to fine tune the amino acid incorporation ratio. The completed LTM or CBM design plan is then sent to the DNA synthesizer, which performs automated synthesis of the primers of oligonucleotides used in generating a mutagenized gene.
A wild type IgG1 gene can be obtained from available sources and amplified by standard techniques (Example 1A). A chimeric surface expression Fc wild type gene construct (approximately 0.65 kb) can be assembled in vitro by SOE-PCR by fusing at the N-terminal, an extracellular export signal and at the C-terminus, a membrane anchoring signal. A list of potential N-terminal extracellular export signals include those from human IgG1 and murine IgGk (SEQ ID:7). The list of potential C-terminal membrane anchoring signals include; placental alkaline phosphatase protein (PLAP), membrane IgM and Platelet Derived Growth Factor (PDGF) (SEQ ID: 8). The various fusion constructs are diagrammatically illustrated in
In some applications it may be desirable that the CH2 domain is proximal to the cell surface membrane and the CH3 is distal (
The Fc-LTM libraries used in the invention are prepared by Kunkel mutagenesis of the Fc expression construct prepared in Section A above, and as detailed in Example 2. A single-stranded Fc template for Kunkel was prepared as in Example 2A. Kunkel mutagenesis of the template was carried out according to standard methods, as detailed, for example, in Kunkel, T. A. (1985) Proc. Natl. Acad. Sci. USA 82:488-92; Kunkel, T. A. et al. (1987) Meth. Enzymol. 154: 367-82; Zoller, M. J. and Smith, M. (1983) Meth. Enzymol. 100:468-500; Hanahan, D. (1983) J. Mol. Biol. 166:557-80; and Maniatis, T., Fritsch, E. F. and Sambrook, J. (1989) in Molecular Cloning, A Laboratory Manual.
In practice, a single reaction scheme such as illustrated in
Double, triple and quadruple regional LTM libraries can be created as above but instead of using the wild type Fc gene as the Kunkel template, a previously generated LTM library template is chosen instead. To create a double LTM library for both “contact” sub regions 1 and 3, previously generated LTM “contact” sub region 1 mutant genes are used as single stranded templates to which are annealed a set of sub region 3 oligonucleotides to generate the double LTM library. The double LTM library can then be used as templates to incorporate LTM “contact” sub region 4 oligonucleotides to make the triple LTM libraries. By progressively utilizing the starting single and double LTM libraries, more complex arrays of LTM library can be developed using all the iterations of the LTM amino acids (
Prior to the Kunkel LTM mutagenesis, the Fc domain may be modified to introduced a stop codon into the reading frame in the various sub-regions to be examined by LTM. For example in regional Fc-FcγRIIIa “contact” point LTM library, there are four separate “stop-modified” templates. The wild type Fc template was “stop-modified” using the oligonucleotides shown in SEQ ID: 28. The purpose is that a “stop-modified” wild type template, which did not undergo Kunkel mutagenesis, will be expressed as an N-terminal truncated protein. These truncation constructs will be composed of an extracellular signal leader and varying lengths of the Fc domain. However, translation of the non-mutagenized reading frame will not continue through to the trans-membrane anchoring signal. Therefore, the “stop-modified” templates will be translated, exported but will not be retained on the extracellular cell surface (comparing
These “stop modified” templates allow a supplementary feature of re-introducing the wild type coding sequence. The addition of “open reading frame” oligonucleotides (SEQ ID: 29) allows the stop codon to be replaced with the original Fc codon. In this manner, the “wild type” re-introduction mutagenesis is proportional to that being introduced by the LTM oligonucleotides. The Fc-LTM surface expression libraries will therefore have an internal wild-type reference control that is not in relative overabundance.
Once the Fc template is LTM modified, the construct is excised from the cloning vector, purified, and ligated into a suitable expression vector (e.g., Clontech, Palo Alto, Calif.). Following E. coli transformation and selection on LBamp plates, the constructs may be sequenced to confirm the Fc desired coding changes and the adjacent extracellular secretion and membrane targeting regions.
A variety of methods for selectable antibody expression and display are available. These include biological “particles (cells or viral particles) such as bacteriophage, Escherichia coli, yeast, and mammalian cell lines. Other methods of antibody expression may include cell free systems such as ribosome display and array technologies which allow for the linking of the polynucleotide (i.e., a genotype) to a polypeptide (i.e., a phenotype) e.g., Profusion™ (see, e.g., U.S. Pat. Nos. 6,348,315; 6,261,804; 6,258,558; and 6,214,553).
One preferred expression system includes' a mammalian cell that is (i) capable of producing clinical-grade monoclonal antibodies, (ii) nonadherent in culture, and (iii) readily transduced with retrovirus. Exemplary cells having these characteristics are BaF3, FDCP1, CHO, and NSO cells.
These cells can be transduced with Fc library expression vectors according to known procedures. In the method detailed in Example 3, an pLXSN mammalian expression vector containing a promoter element, which mediates the initiation of transcription of mRNA, the Fc coding sequence, and signals required for the termination of transcription and polyadenylation of the transcript is transfected into the amphotropic packaging cell line PA317.
This section considers methods for screening the expressed Fc fragments of the above Fc-LTM libraries for enhanced effector function. Subsection A below describes several Fc receptor proteins and indicates for each, desired changes (increases or decreases) in binding affinity that may be screened for. As noted in Section II, this effector function will be related to (i) a shift in binding affinity constant (KD), with respect to a selected IgG1 Fc binding protein, relative to native IgG1 Fc; and/or (ii) a shift in the binding off-rate constant (Koff); with respect to a selected IgG1 Fc binding protein, relative to native IgG1 Fc. Thus, the expressed Fc libraries can be screened for a change in binding constant, which can be an increase or decrease in binding constant, depending on the binding constant being measured, the Fc binding protein involved, and the desired effect of the change in binding constant, as described below in Subsection B. Alternatively, and according to a novel screening method in the invention, the LTM library Fc fragments can be screened directly for an enhanced effector function related to CDC or ADCC, by measuring the extent of cell lysis directly in Fc-expressing cells, as disclosed in Subsection C. Specific receptor targets are given in Subsection D.
This section considers various Fc receptor proteins (targets), and the therapeutic implications of achieving enhanced or reduced Fc binding to the proteins for the four main subclasses of IgG antibodies. Generally, if Fc mediated effector functions are to be enhanced, it is usually desirable to increase binding of IgG1 and IgG3 to those Fc receptors that mediate effector activity, such as the FcγRIIIa receptor. However, some applications require decreased binding to FcγR receptors of any type. For example, those IgGs of all isotypes having Fc fragments conjugated to cytotoxic payloads (radioactive-labels) would otherwise bring healthy FcγR bearing immune cells in to the Fc-radio-conjugates and kill them. In other applications, it may be desirable to have a purely neutralizing antibody that has no effector function. In this circumstance, IgG2 and IgG4 have low affinity to most Fc receptors, but it may be desirable to further reduce Fc receptor binding to these isotypes. For example, IgG4 binding to FcγRI could be further reduced, and IgG2 binding to FcγRIIa could be reduced to minimize effector functions. IgG3 has lower affinity for FcRN, and increasing affinity towards this receptor should increase the circulating half-lives of the antibody.
In the table below an up arrow ↑ is used to indicate an increased affinity of the Fc fragment for the associated Fc binding partner. This increased affinity can be achieved by an increased binding affinity constant KD, or a decreased Koff rate constant. An increased binding affinity constant will reflect a change toward a smaller-valued number, e.g., 10−7 M to 10−8 M. A decreased Koff value will mean a lower-valued Koff, indicating that Fc-binding receptor complex has a reduced tendency to dissociate. Similarly, a down arrow ↓ in the table ↑ is used to indicate a decreased affinity of the Fc fragment for the associated Fc binding partner. This decreased affinity can be achieved by a decreased binding affinity constant KD, or an increased Koff rate constant. A decreased binding affinity constant will reflect a change toward a larger-valued number, e.g., 10−8 M to 10−7 M. An increased Koff value will mean a higher-valued Koff, indicating that Fc-binding receptor complex has a greater tendency to dissociate. A sideways arrow → in the table means no (or substantially no) change in the binding affinity.
Considering the various Fc receptors listed in the table, C1Q is the complement binding complex present in plasma that plays an essential part in CDC, as described above. A target cell recognized by IgG antibody that binds C1q will direct complement mediated cell death (CDC). Increasing C1q affinity for IgG1 and IgG3 will increase CDC function (increasing KD and/or decreasing Koff). Decreasing C1Q affinity for IgG2 (decreasing KD and/or increasing Koff) increasing can reduce unwanted effector activity involving IgG2 antibodies receptor
/↑
/↑
/↑
↑
/↑
↑
/↑
The FcγRI receptor is a high affinity receptor found on monocytes, macrophages, neutrophils and functions in phagocytosis and ADCC. FcγRI has high affinity for IgG1 and IgG3, and increasing the affinity of IgG1 and IgG3 Fc's for FcγRI will increase ADCC function. The natural affinity of FcγRI for IgG2 and IgG4 is none or very low, respectively. Further decreasing the FcγRI affinity of IgG2 and IgG4 Fcs can reduce unwanted receptor, interaction and unwanted effector activity.
FcγRII receptors (FcγRIIa, FcγRIIb, FcγRIIc) are found on B cells, platelets, basophils, eosinophils, neutrophils, monocytes and macrophages, and bind to IgG1 and IgG3 Fc fragments, but bind to IgG2 and IgG4 only weakly or not at all. FcγRIIa/c receptors are positive regulators of Fc functions; FcγRIIb receptor is a negative regulator involved in feedback inhibition of Ig production. Increasing the affinity of IgG1 and IgG3 Fc's for FcγRIIa/c will increase Fc mediated ADCC effector functions. Decreasing the affinity of IgG1 and IgG3 Fc's for FcγRIIb will lessen the feedback inhibition. Further, decreasing the affinity of IgG2 Fc's for FcγRIIa/c will reduce ADCC stimulation of IgG2 isotype. Increasing the affinity of IgG2 and IgG4 Fcs for FcγRIIb will further negatively regulate ADCC activity.
The FcγRIII receptors (FcγRIIIa and FcγRIIIb) are high affinity receptors found on monocytes, macrophages, neutrophils and NK cells and functions in phagocytosis and Antibody Dependent Cellular Cytotoxicity (ADCC). FcγRIIIa is a positive regulator of Fc functions, and FcγRIIIb, a negative regulator as it performs no intracellular signaling. FcγRIII's have affinity for IgG1 and IgG3. Thus, increasing the affinity of IgG1 and IgG3 Fcs for FcγRIIIa will increase Fc mediated ADCC effector functions.
The FcRN receptor functions in the maintenance of constant IgG levels by removing IgG from circulation and recycling through the intracellular vesicles. FcRN has high affinity for IgG1, IgG2 and IgG4 which, through recycling, allows for 3 week circulation ½ life. FcRN has a lower affinity for IgG3 which results in a much shorter circulatory ½ life. Maintaining or increasing the FcRN affinity for IgG1 and IgG3 will thus improve circulation half life of IgGs and promote extended IgG1 and IgG3 effector functions. In certain embodiments, it may be advantageous to have reduced half-lives. For example, it may be undesirable to have circulating radiolabeled antibodies, since it may cause non-specific toxicity to blood cells. Reduced binding to FcRN would allow faster clearance of the unbound radiolabeled antibody.
Protein A is an IgG-binding protein that allows affinity purification of antibodies from cell culture manufacturing. Maintaining or increasing the Protein A affinity for all IgG isotypes would permit better purification from other cellular and growth media components.
Example 4 described methods for obtaining or producing various Fc receptors in soluble form, for use in the screening assays described below for determining KD or Koff values, and where appropriate, biotinylation of the receptor proteins. These include biotinylated Ciq (Example 4A), FcγRIIIa 176V and its polymorphic construct FcγRIIIa 176F, FcγRIIIa 176V, FcγRIIb and the polymorphs of FcγRIIa, FcγRIIIa176F and its polymorphic construct FcγRIIIa176V (Example 4B), and FcR receptor (Examples 4C-4E). BIAcore analysis was carried out to assess the functional IgG Fc binding and the preliminary affinities (KD) of refolded FcγRIIIa fragments, as detailed in Example 5, with reference to
This subsection will describe methods for screening Fc fragments produced by the Fc-LTM libraries for enhanced effector function, based on a desired change (increase or decrease) in either KD or Koff. In either method, it is generally desirable to preselect cells for those expressing functional Fc fragments, that is, cells expressing Fc fragments cable of binding with at least moderate affinity to a selected Fc receptor.
In the pre-selection method illustrated in
The reaction steps involved in the pre-selection method are shown in
The pre-selection method illustrated in
Initially, the Fc-expression cells (typically pre-selected for functional Fc expression), are equilibrated with biotinylated FcγRIIIa, producing a mixture of cells having bound biotinylated FcγRIIIa, and low-affinity and non expressing cells. Following equilibration binding to FcγRIIIa, streptavidin coated beads are added to the mixture, forming a binding complex consisting of high-affinity expressing cells, biotinylated FcγRIIIa, and magnetic beads. The complexes are isolated from the mixture using a magnet, and the bound complex is washed several times under stringent conditions to remove complexes of low-affinity cells and non-specifically bound cells. The resulting purified complexes are released from the complexes, by treatment with a suitable dissociation medium, to yield cells enriched for expression of high-affinity Fc fragment.
In one exemplary screening method, the isolated cells are plated at low density, and clonal colonies are then suspended in medium at a known cell density. The cells are then titrated with biotinylated FcγRIIIa by addition of known amounts of FcγRIIIa, as indicated, e.g, from 10 pM to 1000 nM. After equilibration, the cells are pelleted by centrifugation and washed one or more times to remove unbound FcγRIIIa, then finally resuspended in a medium containing fluoresceinated spreptavidin. The fluoresceinated cells are scanned FACS to determine an average extent of bound fluorescein per cell. The Fc fragments selected will having a binding affinity that is preferably at least 1.5 higher, and typically between 1.5-2.5 higher (or lower, if decreased binding affinity is desired) than that of wildtype Fc fragments with respect to the selected receptor.
Alternatively, the Fc fragments expressed on the expression cells may be selected for enhanced Koff, i.e., a lower-valued Koff, where increased binding affinity is desired, or a higher-valued Koff, where reduced binding affinity is desired. The Fc fragments selected will preferably have Koff values that are at least 1.5 and up to 2-5 fold lower than the measured Koff for wildtype Fc fragment, when measured under identical kinetic binding conditions (or 1.5 to 2.5 fold higher if lower affinity Fc fragments are sought).
In the method for determining Koff values, Fc-expressing cells are incubated with a saturating amount of biotinylated Fc receptor, e.g., biotin-labeled FcγRIIIa, under conditions, e.g., 30 minutes at 25° C., with shaking, to effectively saturate displayed Fc fragment with bound receptor. The cells are then incubated with non-biotinylated FcγRIIIa at saturating conditions, for a selected time sufficient to reduce the percentage of biotinylated FcγRIIIa bound to the cells as a function of the off rate of the antigen. Following incubation, the cells are centrifuged, and washed to remove unbound biotinylated FcγRIIIa, yielding cells which contains a ratio of biotinylated and native FcγRIIIa in proportion of the antibody's Koff.
Details of the method are given in Example 7.
The koff values are then determined by incubating the cells with a fluoresceinated streptavidin (streptavidin-PE) and a fluoresceinted cell marker (anti-his-fluorescein), washing the cells, and sorting with FACS. The koff value is determined from the ratio of the two fluorescent markers, according to known methods. Example 7 provides additional details for the method.
In some cases, it may be advantageous to select Fc fragments having enhanced binding affinity for one Fc receptor and altered, e.g., decreased binding activity for a second Fc receptor.
After performing the binding affinity assay, those cells exhibiting a desired enhancement in Fc characteristics can be expanded for growth expansion. The Fc-LTM sequence from these clones are then “rescued” by PCR with Fc-LTM vector specific primers and subcloned into a suitable sequencing vector for sequence analysis and identification of the LTM amino acid change. Enhanced activity clones (either increased or reduced binding affinity with respect to a particular Fc receptor) thus identified may be further tested for actual effector function, e.g., in a CDC or ADCC assay of the type described below.
Exemplary receptors targets, and desired enhancement in binding affinity include one of: (i) an elevated binding affinity constant or reduced binding off-rate constant, with respect to Fc-binding protein C1q, FcγRI, FcγRIIa, and FcγRIIIa, (ii) a reduced binding affinity constant or elevated binding off-rate constant with respect to Fc-binding proteins FcγRIIb, FcγRIIIb; and an elevated or reduced binding affinity constant or a reduced or elevated binding off-rate constant, respectively, with respect to Fc-binding protein FcRN and protein A.
For some experiments, the method was used to monitor the quantitative ADCC effector differences in between individuals with either FcγRIIIa F158/V158 and/or FcγRIIa H131/R131 polymorphisms, as detailed in Experiment 9.
After the LTM Fc variants are screened and selected using functional assays, the rescue of those clones then allows for identification of that DNA coding sequences. In the combinatorial beneficial mutation (CBM) approach, coding sequences are subsequently generated which represent combinations of the beneficial LTM mutations identified and combines them together into a single library. These combinations may be combinations of different beneficial mutations within a single sub-region or between two or more sub-region within the Fc. Therefore, synergistic effects of multiple mutations can be explored in this process.
The combinatorial approach resembles the Walk Through Mutagenesis method (U.S. Pat. Nos. 5,798,208, 5,830,650, 6,649,340B1 and US20030194807) except that the selected codon substitutions within the Fc sub regions are the different beneficial amino-acid substitutions identified by LTM. As shown in
In accordance with one aspect of the invention, desired enhancements in effector function related to enhanced or inhibited CDC or ADCC can be screened directly, using Fc-expressing cells as the target cells for the screen. The method will be described with reference to
In the direct screening procedure, detailed in Example 8A and 8B, a pre-selected library obtained as above is diluted, and individual clonal cells placed in the wells of a microtitre plate, with a second “replica” plate being formed with the same cells. Human serum complement, including the C1q complex, is prepared as in Example 8B and added in serial dilutions to the microtitre plate wells, and the resulting CDC activity is measured fluorometrically. Those cells showing highest CDC levels, expressed in terms of amount complement added, may be identified as having a desired enhanced CD effector function, and/or may be expanded and re-screened for CDC activity until cells exhibiting a desired enhancement in CDC activity are identified. As above, when enhanced Fc fragments are identified, the associated cell-expression vectors can be analyzed to determine the Fc-coding sequence of the fragment.
The mechanism of cell lysis in of antibody-dependent cellular cytotoxicity ADCC), is illustrated in
In the direct screening procedure, detailed in Example 8C, a pre-selected library obtained as above is diluted and individual clonal cells placed in the wells of a microtitre plate, with a second “replica” plate being formed with the same cells. To the microtitre plate wells are is added PBMCs including NK cells having surface-expressed receptor. After incubation, the cells are centrifuged and the cell supernatant assayed for released LDH, as detailed in Example 8C. Those cells showing highest levels of ADCC activity may be selected for enhanced Fc activity, and/or may be expanded and rescreened for ADCC activity until cells showing a desired enhancement in ADCC are identified.
After performing the Fc effector cell assays, those corresponding replica daughter wells exhibiting the desired level of ADCC or CDC activity can be expanded for growth expansion. The Fc-LTM sequence from these clones are then “rescued” by PCR with Fc-LTM vector specific primers and subcloned into a suitable sequencing vector for sequence analysis and identification of the LTM amino acid change.
After identification and sequencing of enhanced affinity Fc fragments, the identified sequences can be used, for example, in the construction of full length antibodies or single-chain antibodies having a selected antigen-binding specificity and an enhanced receptor function, e.g., an ability to enhance or suppress CDC or ADCC when administered to a subject, as discussed above. Example 10 described the construction of a full-length Rituxin antibody having enhanced CDC or ADCC function.
The following examples illustrate, without limitation, various methods and applications of the invention.
The wild type IgG1 was obtained from (image clone #4765763, ATCC Manassas, Va.). The amino acid and DNA sequences of the individual CH2 and CH3 domains are shown in SEQ IDs:1-4 respectively. The IgG1 Fc gene (SEQ ID:5 and 6) was PCR amplified and cloned into pBSKII (Stratagene, La Jolla, Calif.) for propagation, miniprep DNA purification and production of single stranded DNA template (QIAgen, Valencia Calif.).
Fc domain PCR reactions were performed using a programmable thermocycler (MJ Research, Waltham, Mass.) and comprised of; Forward Fc PCR primer 5′-TAT GAT GTT CCA GAT TAT GCT ACT CAC ACA TGC CCA CCG T-3′, Reverse Fc PCR primer 5′-GCA CGG TGG GCA TGT GTG AGT AGC ATA ATC TGG AAC ATC A-3′, 5 μl of 10 uM oligonucleotide mix, 0.5 μl Pfx DNA polymerase (2.5 U/μl), 5 μl Pfx buffer (Invitrogen, Calsbad, Calif.), 1 μl 10 mM dNTP, 1 μl 50 mM MgSO4 and 37.5 μl dH20 at 94° C. for 2 min, followed by 24 cycles of 30 sec at 94° C., 30 sec at 50° C., and 1 min at 68 C and then incubated for a 68° C. for 5 min.
The chimeric surface expression Fc wild type gene construct (approximately 0.65 kb) was assembled in vitro by SOE-PCR by fusing at the N-terminal, an extracellular export signal and at the C-terminus, a membrane anchoring signal. A list of potential N-terminal extracellular export signals include those from human IgG1 and murine IgGk (SEQ ID:7). The list of potential C-terminal membrane anchoring signals include; placental alkaline phosphatase protein (PLAP), membrane IgM and Platelet Derived Growth Factor (PDGF) (SEQ ID: 8). The various fusion constructs are diagrammatically illustrated in
The PCR products; N-terminal leader signal, Fc gene, and C-terminal membrane anchor section were then all incubated together as a mixture (5 μl of 10 uM oligonucleotide mix) and assembled by SOE-PCR using 0.5 μl Pfx DNA polymerase (2.5 U/μl), 5 μl Pfx buffer (Invitrogen), 1 μl 10 mM dNTP, 1 μl 50 mM MgSO4 and 37.5 μl dH20 at 94° C. for 2 min, followed by 24 cycles of 30 sec at 94° C., 30 sec at 50° C., and 1 min at 68° C. and then incubated at 68° C. for 5 min. The SOE-PCR assembly reaction permitted oligonucleotide overlap annealing, base-pair gap filling, and ligation of separate DNA fragments to form a continuous gene. The Fc DNA from the PCR reaction was then extracted and purified (Qiagen PCR purification Kit) for subsequent Xho I and EcoRI restriction endonuclease digestion as per manufacturer's directions (New England Biolabs, Beverly Mass.). The chimeric Fc surface expression construct was then subcloned into pBSKII vector and sequenced to verify that there were no mutations, deletions or insertions introduced. Once verified, this chimeric N-terminal leader signal, Fc gene, and C-terminal membrane anchor surface expression construct served as the wild type template for the subsequent strategies of building Fc-LTM libraries.
Various Fc surface expression constructs (
In some applications it may be desirable that the CH2 domain is proximal to the cell surface membrane and the CH3 is distal (
All the above Fc expression constructs were cloned in PBSKII for the preparation of Fc single stranded DNA. The E. coli hosts CJ236 were grown in 2YT/Amp liquid medium until the OD600 reached approximately 0.2 to 0.5 Absorbance Units. At this timepoint, 1 mL of M13 K07 helper phage was added to the bacterial culture for continued incubation at 37° C. After 30 minutes, the bacteria and phage culture was transferred to a larger volume of 2YT/Amp liquid medium (30 mL) containing 0.25 ug/mL Uridine for overnight growth.
The next day, the culture medium was clarified by centrifugation (10 min at 10000 g) after which the supernatant was collected and 1/5 volume of PEG-NaCl added for 30 minutes. The mixture was further centrifuged twice more but after each centrifugation, the supernatant was discarded in favor of the retained PEG/phage pellet. The PEG/phage pellet was then resuspended in PBS (1 mL), re-centrifuged (5 min at 14000 g). The supernatant was collected and then applied to DNA purification column (QIAprep Spin M13, Qiagen) to elute single stranded wild type IgG1 Fc uridinylated-DNA.
Synthetic oligonucleotides were synthesized on the 3900 Oligosynthesizer (Syngen Inc., San Carlos, Calif.) as per manufacturer directions and primer quality verified by PAGE electrophoresis prior to PCR or Kunkel mutagenesis use. LTM analysis introduces a predetermined amino acid into every position (unless the wildtype amino acid is the same as the LTM amino acid) within a defined region (US2004020306). In contrast to other stochastic mutagenesis techniques, the LTM oligonucleotide annealed to uridinylated single stranded template and is designed to mutate only one defined Fc amino acid position.
As described in the specification above, there are two Fc libraries constructed for LTM analysis. The first embodiment is being termed an “unbiased” CH2×CH3 library where each amino acid position in the Fc region will be replaced by the nine chosen LTM amino acids (
The second Fc LTM library represents the four separate IgG1 Fc-FcγRIIIa “contact” points as identified from the IgG1 Fc-FcγRIIIa co-crystal structure (
In the example of the “unbiased” CH2×CH3 library, five glycine LTM replacement oligonucleotides (SEQ ID:27) are used to perform similar substitutions of at the first sub-region of the CH2 domain defined by the amino acid sequence LLGGPSV (SEQ ID: 12).
The pLXSN mammalian expression vector contains one promoter element, which mediates the initiation of transcription of mRNA, the polypeptide coding sequence, and signals required for the termination of transcription and polyadenylation of the transcript. pLXSN contains elements derived from Moloney murine leukemia virus (MoMuLV) and Moloney murine sarcoma virus (MoMuSV), and is designed for retroviral gene delivery and expression.
Briefly, the pLXSN/Fc construct is transfected into the amphotropic packaging cell line PA317 (or other alternative cells) by calcium phosphate precipitation (Gibco, Carlsbad, Calif.).
The ecotropic cell line pECO (Clontech) is grown in Growth Medium (DME containing 10% heat inactivated fetal bovine serum, 100 U/ml Penicillin, 100 U/ml Streptomycin, 2 mM L-Glutamine). The following procedure is illustrated in
For suspension cells such as NS0, a mouse myeloma cell line with lymphoblastic morphology, the cells are grown to log phase growth to approximately 5×105 cells/ml. The NS0 cells are pelleted after a brief centrifugation and resuspended in 1 ml of fresh media containing diluted retroviral supernatant (>100 folds) and incubate for 12-24 hours at 37° C. A series of test dilutions can be performed with the retroviral supernatant to optimize transduction efficiency. NS0 library cells can then be monitored for transduction efficiency and Fc-LTM expression by subsequent FACS analysis.
Murine tumor cell line NS0 is transduced with the harvested pLXSN/Fc retroviral vector supernatants (transient system shown in
The essential goal in our screening process is for each mammalian cell to express LTM Fc-fusion protein on its cell surface. Surface expression of Fc can be determined by anti-human anti-Fcγphycoerytherin antibody, or by also staining for the Myc or HA tags (all PharMingen, San Diego, Calif.) and confirmed by flow cytometry. pLXSN/Fc NS0 transduced cells are collected by low speed centrifugation (5 mins at 500 g), washed twice with CSB (PBS and 0.5% BSA), resuspended, and then incubated with soluble anti-Fcγ-PE antibody. After 1 hour (in the dark, covered and on ice) the cells are twice washed with cold CSB and resuspended at a concentration of 10×106 cells/mL. Negative control cells are NS0 transduced with empty pLXSN vector and positive control cells are pLXSN with wild type Fc. The pLXSN-Fc transformed cells should show a significant shift in fluorescence, compared to empty pLXSN vector. The cells are then analyzed on FACSscan (Becton Dickinson) using CellQuest software as per manufacturer's directions.
After Fc surface expression on the LTM library cells is confirmed, the next task is to verify that the extracellular Fc constructs are capable of binding Fc receptors, namely FcγRIIIa and C1q. This is essential as the initial pre-selection procedures and subsequent Fc effector functional assays require Fc receptor association. To investigate, NS0 cells expressing the wild type Fc domain are collected as above and incubated with either labeled FcγRIIIa or C1q protein. The FcγRIIIa or C1q proteins can be either phycoerytherin or FITC fluorescently labeled or biotinylated as described below. For example, NS0 cells expressing Fc variants capable of binding biotin-C1q can then be counterstained with secondary streptavidin-PE and analyzed by FAGS. Functional FC-LTM variants will bind the labeled FcγRIIIa and/or C1q protein and yield higher fluorescence readings. The protocols below describe the procedures to isolate, purify and biotin label FcγRIIIa or C1q proteins.
Bioactive C1q protein is composed as a heterotrimer [SEQ ID:30-32] and available commercially in a purified form (Calbiochem, San Diego, Calif.). Biotinylation of the C1q protein can be accomplished by a variety of methods however; over-biotinylation is not desirable as it may block the epitope-antibody interaction site. The protocol used was adapted from Molecular Probes FluoReporter Biotin-XX Labeling Kit (cat# F-2610). Briefly, C1q 1 μl of 0.9 mg/ml stock (Calbiochem), was added to 100 μM sodium bicarbonate Buffer at pH 8.3 and 9.4 μl of Biotin-XX solution (10 mg/ml Biotin-XX solution in DMSO). The mixture was incubated for 1 hour at 25° C. The solution was transferred to a micron centrifuge filter tube, centrifuged and washed repeatedly (four times) with PBS solution. The biotinylated-C1q solution was collected, purified over a Sephadex G-25 column, and the protein concentration determined by OD 280.
The DNA sequence of FcγRIIIa176V was obtained from ATCC (SEQ ID: 33). The FcγRIIIa176F polymorphism construct was re-engineered by Kunkel mutagenesis as described above (SEQ ID: 34). The following E. coli purification protocol also pertains to the extracellular domain of FcγRIIb (SEQ ID: 35 and 36) and FcγRIIa (SEQ ID: 40, 41 and 42). FcγRIIIa176F and FcγRIIIa176V were cloned into pET 20b expression vector (Invitrogen, Carlsbad, Calif.) which appended a C-terminal 6×HIS tag to the protein. The pET 20b-FcγRIIIa V/F176 constructs were then transformed into BL21 E. coli host cells. Liquid cultures (LB-Amp) of E. coli cells were expanded from overnight small scale (5 mL) to 250 (mL) and upon reaching an absorbance value of (0.5 @600 nm) the FcγRIIIa protein was induced with IPTG (0.5 mM) for 4 hours at 25° C. If not immediately used in the following purification scheme, growth cultures were subsequently pelleted and stored at −80° C. Cell pellets were then resuspended in 6 ml B-PER® II lysis Reagent (Pierce, Rockford, Ill.) by vigorous vortexing until they were without large visible aggregate clumpings. Once uniformly suspended, the cells were gently shaken at RT for 10 minutes. After which, the cell lysis mixture was centrifuge (10 min at 10000 RPM) to initially separate soluble proteins from the insoluble proteins. The extracellular domains of the FcγFIIa H/R131 polymorphisms were cloned in the same fashion.
The lysis supernatant was (collected and saved/discarded) while the pellet was again resuspended in 6 ml B-PER® II reagent. Lysozyme was added to the resuspended pellet at a final concentration of 200 □g/ml and incubated at RT for 5 minutes. The insoluble inclusion bodies were then collected by centrifugation (30 min at 10000 RPM). The resulting pellet was again resuspended in 15 ml of B-PER® II (approximately 1:20 pellet volume to B-PER dilution) and mixed by vigorous vortexing. The inclusion bodies were collected by centrifugation (15 min at 10000 RPM). The steps of pellet resuspension, vortexing and centrifugation were repeated ten more times after which the final pellet of the purified inclusions bodies was saved and stored.
Purified inclusion body was thawed on ice and resuspended in 1.5 ml Buffer B [100 mM NaH2PO4, 10 mM Tris Cl, 8 M Urea, pH: 8]. Taking care to avoid foaming, the suspension was slowly stirred for approximately 60 minutes (RT) or until lysis is completed (as observed when the solution becomes translucent). The mixture was centrifuged (15 min at 10000 RPM) to pellet the cellular debris. The supernatant (cleared lysate) was then collected and added to it, 5 mL of Ni-NTA resin (Qiagen) and mixed gently (60 minutes at 4° C.). The lysate-resin mixture was carefully loaded into an empty column and wash with 100 ml Buffer B (pH: 6.3). The recombinant protein was then eluted with 20 ml Buffer B (pH: 4.5).
The Ni-NTA purified FcR protein, 3 mL from above, was added dropwise with stirring to refolding buffer [0.1 M Tris/HCl, 1.4 M arginine, 150 mM NaCl, 5 mM reduced glutathione, 0.5 mM oxidized glutathione, 0.1 mM phenylmethylsulfonyl fluoride, 0.02% NaN3} over a 6 hour time period and then stirred for 72 hours. The renatured protein solution was then dialyzed against 4 L of dialysis buffer [0.1 M Tris/HCl, 5 M NaCl, 0.1 M MgCl2.6H2O] that was replaced with fresh buffer twice more before an overnight dialysis period. Ni-NTA resin (2 mL) was added to the renatured protein solution and then gently stirred for 60 minutes (RT). The lysate-resin mixture was carefully loaded into an empty column and wash with 100 ml wash buffer B (10 mM Tris/HCl, 300 mM NaCl, 50 mM imidazole, pH: 8.0). The recombinant protein was then eluted with 10 ml elution buffer (10 mM Tris/HCl, 300 mM NaCl, 250 mM imidazole, pH: 8.0).
To assess functional IgG Fc binding and gauge the preliminary affinities (KD=kd/ka=koff/kon) of the refolded FcγRIIIa fragments, BIAcore—2000 surface plasmon resonance system analysis was employed (BIAcore, Inc. Piscatawy, N.J.). The ligand, human full length IgG1 (Calbiochem) was immobilized on the BIAcore biosensor chip surface by covalent coupling using N-ethyl-N′-(3-dimethylaminopropyl)-carbo-diimide hydrochloride (EDC) and N-hydroxsuccinimide (NHS) according to manufacturer's instructions (BIAcore, Inc). A solution of ethanolamine was injected as a blocking agent.
For the flow analysis, FcγRIIIa was diluted in BIAcore running buffer (20 mM Hepes buffered Saline pH 7.0) into three concentrations of 0.13 □M, 0.26 □M, and 0.52 □M. The aliquots of FcγRIIIa were injected at a flow rate of 2 □l/minute for kinetic measurements. Dissociation was observed in running buffer without dissociating agents. The kinetic parameters of the binding reactions were then determined using BIAevaluation 2.1 software.
We have also measured the koff kinetic difference between FcγRIIIaV158 and the Fc□RIIIaF158 polymorphisms and are shown in the table below. These preliminary results are in agreement with other publications where the FcγRIIIaF158 polymorphism has lower affinity to IgG1 Fc as demonstrated by a six-fold faster koff kinetic.
After growth culture, the NS0 Fc-LTM cells are labeled by incubating with biotinylated C1q at saturating concentrations (400 nM) for 3 hours at 37° C. under gentle rotation. To remove unbound biotinylated C1q, NS0 cells are then washed twice with RPMI growth medium before being resuspended 1.0×105 cells/μl in PBS. A ratio of single cell suspension of approximately 107 cells (100 μl) is mixed with 10 μl streptavidin coated or anti-biotin microbeads (MACS, Miltenyi Biotec) is incubated on ice for 20 minutes with periodic inversions. After low speed centrifugation, the mixture is then twice washed with buffer and resuspended in 0.5 mL. These procedures and cellular components are diagrammed in
The cell suspension is applied to a LS MACS column placed in the magnetic field separator holder. The MACS column is then washed with 2×6 mL of buffer removing any unbound cells in the flow-through. The MACS column is then removed from the separator and placed on a suitable collection tube. 6 mL of buffer is loaded onto the MACS column and immediately thereafter, the bound Fc-LTM cells are flushed out through applying the column plunger. Low affinity or non-functional binding Fc-LTM variant cells are not retained in this manner.
This positive selection then recovers only those Fc-LTM variant cells with functional affinity to C1q/FcgRIIIa. This MACS enrichment step will eliminate the need of the FACS to process and sort unwanted cells. After elution, the enriched NS0 cells are then incubated for further culture (
The following methodology involves FACS screening LTM Fc libraries for enrichment and isolation of FcR binding affinity variants. After growth culture, the above NS0 cells are incubated with biotinylated C1q at saturating concentrations (400 nM) for 3 hours at 37 C under gentle rotation. (As before, biotinylated FcγRIIIa can be substituted for those appropriate experiments.) The NS0 cells are then twice washed with RPMI growth medium to remove unbound biotinylated C1q/FcγRIIIa. The cells are then sorted on FACS-Vantage (Becton Dickinson) using CellQuest software as per manufacturer's directions.
Depending on the binding characteristics desired, the sort gate and be adjusted to collect that fraction of the Fc-LTM population. For example, if enhanced affinity for FcγRIIIa is desired, the gate will be set for higher florescence signals. We have shown that FACS gating is able to enrich, by more than 80%, for a higher affinity sub-population in test system with other cell lines and associated binding proteins (
The following studies are performed to demonstrate that surface expression of Fc-LTM by NS0 cells that can lead to the engagement of FcγR on effector cells, such as monocytes and activated granulocytes, thereby initiating FcγR-dependent effector functions (
The FACS pre-sorted library is diluted into 96 well plates. Alternatively, after pLXSN/Fc transduction of NS0 cells, if only a small library is made (106), these cells could also be directly plated at dilution of a single clone/well. These single clone wells can be then grown and expanded into daughter plates. One of these daughter plates can later serve as an Fc-effector assay plate. Thus, in some cases a small Fc-LTM library will not need the above MACS and/or FACS pre-sort.
It should be noted that in the following selection assays for higher affinity to Fc receptors C1q/FcγRIIIa and associated enhanced Fc effector C1q/FcγRIIIa functions, the additional step of screening for lower affinity to other Fc receptors such as FcγRIIb and diminished Fc effector functions can be performed in parallel (
Normal human mononuclear cells were prepared from heparinized bone marrow samples by centrifugation across a Ficoll-Hypaque density separation gradient. Human AB serum (Gemini Bioproducts, Woodland, Calif.) was used as the source of human complement. The ability of the NS0 library cells to promote complement mediated cytotoxicity was measured in an analogous manner. Briefly, the NS0 cells were cultured as above and plated (5×104) were placed in 96-well flat-bottom microtiter wells. Human serum complement (Quidel, San Diego, Calif.) was serially diluted to first gauge a working range of lysis. The mixture of diluted complement and NS0 cell suspensions is then incubated fort h at 37° C. in a 5% CO2 incubator to facilitate CDC. Afterwards, 50 μl of Alamar Blue (Accumed International, Westlake, Ohio) is added to each well and further incubated overnight at 37° C. Using a 96-well fluorometer, the fluorescence reading with excitation at 530 nm and emission at 590 nm is measured.
Typically, the results are expressed in relative fluorescence units (RFU) in proportion to the number of viable cells. The activity of the various mutants is then examined by plotting the percent CDC activity against the log of Ab concentration (final concentration before the addition of Alamar Blue). The percent CDC activity was calculated as follows: % CDC activity=(RFU test−RFU background)×100 (RFU at total cell lysis−RFU background).
Effector PBMCs are prepared from heparinized whole venous blood from normal human volunteers. The whole blood is diluted with RPMI (Life Technologies, Inc.) containing 5% dextran at a ratio of 2.5:1 (v/v). The erythrocytes are then allowed to sediment for 45 minutes on ice, after which the cells in the supernatant are transferred to a new tube and pelleted by centrifugation. Residual erythrocytes are then removed by hypotonic lysis. The remaining lymphocytes, monocytes and neutrophils can be kept on ice until use in binding assays. Alternatively, effector cells can be purified from donors using Lymphocyte Separation Medium (LSM, Organon Technika, Durham, N.C.).
Target NS0 library cells expressing Fc variants are washed three times with RPMI 1640 medium and incubated with purified FcR (all types) at 1 mg/ml (concentration to be determined for maximum ADCC) for 30 min at 25° C. The above purified PBMC effector cells are washed three times with medium and placed in 96-well U-bottom Falcon plates (Becton Dickinson). To first gauge the working range of ADCC for these experiments, three-fold serial dilutions from 3×105 cells/well (100:1 effector/target ratio) to 600 cells/well (0.2:1) are plated. Typically, ADCC is assayed in the presence of 50 fold excess of harvested PMBC.
Target NS0 cells are then added to each well at 3×103 cells/well. Spontaneous release (SR, negative control) is measured by NS0 target wells without added effector cells; conversely, maximum release (MR, positive control) is measured by adding 2% Triton X-100 to NS0 target cell wells. After 4 h of incubation at 37° C. in 5% CO2, ADCC assay plates are centrifuged. The supernatant are then transferred to 96-well flat-bottom Falcon plates and incubated with LDH reaction mixture (LDH Detection Kit, Roche Molecular Biochemicals) for 30 min at 25° C. The reactions are then stopped by adding 50 ml of 1 N HCl. After which, the samples are measured at 490 nm with reference wavelength of 650 nm. The percent cytotoxicity was calculated as [(LDH releasesample−SReffector−SRtarget)/(MRtarget−SRtarget)]×100. For each assay, the percent cytotoxicity versus log(effector/target ratio) is plotted and the area under the curve (AUC) calculated. The assays are performed in triplicate.
For some experiments, as explained in the detailed description, we require monitoring the quantitative ADCC effector differences in between individuals with either FcγRIIIa F158/V158 and/or FcγIIa H131/R131 polymorphisms. There are several ways to genotype the polymorphisms including; PCR followed by direct sequencing, PCR using allele specific primers, or PCR followed by allele-specific restriction enzyme digestion. For our purposes, the latter allele-specific restriction enzyme digestion procedure for FcγRIIIa F158/V158 is described and the methodology is similar for FcγRIIa H131/R131 polymorphism (albeit using different PCR amplification primers).
Genotyping of the FcγRIIIA-158V/F polymorphism is performed by means of PCR-based allele-specific restriction analysis assay. Two FcγRIIIa gene-specific primers: 5′-ATA TTT ACA GAA TGG CAC AGG-3′; antisense SEQ ID: 5′-GAC TTG GTA CCC AGG TTG AA-3′; are used to amplify a 1.2-kb fragment containing the polymorphic site. This PCR assay was performed in buffer with 5 ng of genomic DNA, 150 ng of each primer, 200 μmol/L of each dNTP, and 2 U of Taq DNA polymerase (Promega, Madison, Wis.) as recommended by the manufacturer. The first PCR cycle consisted of 10 minutes denaturation at 95° C., 1½ minute primer annealing at 56° C., and 1½ minute extension at 72° C. This was followed by 35 cycles in which the denaturing time was decreased to 1 minute. The last cycle is followed by 8 minutes at 72° C. to complete extension. The sense primer in the second PCR reaction contains a mismatch that created an NIaIII restriction site only in FcγRIIIA-158V-encoding DNA: 5′-atc aga ttc gAT CCT ACT TCT GCA GGG GGC AT-3′; uppercase characters denote annealing nucleotides, lowercase characters denote nonannealing nucleotides), the antisense primer was chosen just 5′ of the fourth intron: 5′-acg tgc tga gCT TGA GTG ATG GTG ATG TTC AC-3′). This second PCR reaction is performed with 1 μL of the first amplified fragment, 150 ng of each primer, 200 μmol/L of each dNTP, and 2 U of Taq DNA polymerase, diluted in the recommended buffer. The first cycle consisted of 5 minutes' denaturing at 95° C., 1 minute primer annealing at 64° C., and 1 minute extension at 72° C. This was followed by 35 cycles in which the denaturing time was 1 minute. The last cycle was followed by 9½ minutes at 72° C. to complete extension. The 94-bp fragment was digested with NIaIII, and digested fragments were electrophoresed in 10% polyacrylamide gels, stained with ethidium bromide, and visualized with UV light.
FcγRIIa genotyping was determined using gene-specific sense: 5′-GGA AAA TCC CAG AAA TTC TCG C-3′; antisense SEQ ID: 5′-CAA CAG CCT GAC TAC CTA TTA CGCG GG-3′ primers. The sense primer is from the exon encoding the second extracellular domain upstream of codon 131 and ends immediately 5′ to the polymorphic site. It contains a one nucleotide substitution which introduces a Bst UI site (5′˜CGCG-3′) into the PCR product when the next nucleotide is G, but not when the next nucleotide is A. The antisense primer is located in the downstream intron and contains a two nucleotide substitution which introduces an obligate Bst UI site into all PCR products which use this primer. The PCR conditions were as follows: one cycle at 96° C. for five minutes, 35 cycles at 92° C. for 40 seconds and 55° C. for 30 seconds, and one cycle at 72° C. for 10 minutes. Products were digested using Bst UI, which cuts once in the presence of the R131 allele and twice in the presence of the H131 allele. Fragments were resolved by electrophoresis on a 3% agarose gel.
CBM-Fc or LTM-Fc variants that exhibit the desired in vitro Fc receptor binding properties will then be tested for correlative Fc effector functions. For these assays we will compare the CBM-Fc or LTM-Fc variant with the Rituxin Fc to determine if there are differences in ADCC and CDC activity. Developed for the treatment of non-Hodgkin's lymphoma, Rituxin is a chimeric monoclonal IgG1 antibody specific for the B-cell marker CD20. For our purposes, we will compare wild type Rituxin (having the wild type IgG1 Fc region) with chimeric Rituxin (CH1: VH and VL) and CBM-Fc or LTM-Fc variant (hinge, CH2 and CH3) replacement.
By PCR with appropriate primers, the hinge, CH2 and CH3 will be amplified from CBM-Fc or LTM-Fc variant. The primers will also introduce restriction sites into heavy-chain hinge and CH3C-terminus for subsequent restriction digest and cloning. The Rituxin vector has been modified with similar restriction sites at the heavy-chain hinge region and CH3C-terminus without changing to the amino-acid sequence. The modified Rituxin vector then allows simple replacement of the Fc domain while retaining its' VH and VL specificity for CD20.
After sequence verification, the Rituxin-Fc-LTM construct is re-cloned into PcDNA3 vector (Invitrogen) for expression as a soluble IgG1. Briefly, the PcDNA3-Rituxin-Fc-LTM is transfected into CHO-K1 cells using lipofectamine (Invitrogen) and cultured in Dulbecco's modified Eagle's medium with 5% heat-inactivated fetal calf serum. If stable transfected clones are desired, they can then be selected with in the DMEM growth media with supplemented G418 (400 ug/ml). The supernatants from the above transfection are then collected, clarified by centrifugation to pellet all detached cells and debris. The secreted full length Rituxin-Fc-LTM IgG1 can be purified by passing the culture supernatant over a Protein A Sepharose 4B affinity column. After washing with two to three column volumes of PBS, bound Rituxin-Fc-LTM IgG1 protein is eluted with KSCN (3 M) in phosphate-buffered saline (10 mM sodium phosphate, 0.154 M NaCl, pH 7.3). Protein concentrations are estimated using absorbance at 280 nm and can be stored long term in phosphate-buffered saline (pH 7.3), containing sodium azide (0.8 mM) at −20° C.
The purified antibody is then added to WILS-2 target cells for ADCC, CDC or apoptosis assays. Apoptosis of WIL2-S cells can be analyzed by flow cytometric analysis using propidium iodide (PI; Molecular Probes, Eugene, Oreg.) and annexin V-FITC (Caltag, Burlingame, Calif.). Briefly, 5×105 WIL2-S cells are incubated with the specified concentrations of Rituxin wild type or Rituxin grafted Fc-LTM for 24 h at 37° C. and 5% CO2. The target WIL2-S cells are then washed in PBS and resuspended in 400 ml of ice-cold annexin binding buffer (BD PharMingen, San Diego, Calif.) to which 10 ml of annexin V-FITC and 0.1 mg PI are added. Cells are then analyzed on a flow cytometer (Beckman-Coulter, Miami, Fla.): for excitation at 488 nm and measured emission at 525 nm (FITC) and 675 nm (PI) after compensation for overlapping emission spectra.
Although the invention has been described with respect to particular embodiments and applications, it will be appreciated that various modification and changes may be made without departing from the invention.
Number | Date | Country | |
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60675345 | Apr 2005 | US |
Number | Date | Country | |
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Parent | 11912568 | Sep 2008 | US |
Child | 13268149 | US |