Antibodies that modulate a biological activity expressed by a cell

Abstract

The invention provides means and methods for interfering with Programmed Cell Death 1 protein (PD-1) and Lymphocyte activation 3 (LAG 3) mediated inhibition in a PD-1 and/or LAG3 positive cell. A method may comprise contacting said cell with an antibody or a functional part, derivative and/or analogue thereof that comprises a variable domain that can bind to an extracellular part of PD-1 and a variable domain that can bind to an extracellular part of LAG3, thereby inhibiting PD-1 and/or LAG3 mediated activity in said cell. The invention also provides antibodies or variants thereof that comprises a variable domain that can bind to an extracellular part of PD-1 and a variable domain that can bind to an extracellular part of LAG3.

Claims

1. An antibody or variant thereof that comprises a variable domain that can bind to an extracellular part of PD-1 and a variable domain that can bind to an extracellular part of LAG3; wherein the variable domain that can bind to an extracellular part of PD-1 blocks the binding of PD-1 to PD-L1 and/or PD-L2; and wherein the variable domain that can bind to an extracellular part of LAG3 comprises a heavy chain variable region with a CDR1, CDR2 and CDR3 region that comprises the amino acid sequence of the CDR1, CDR2 and CDR3 of a heavy chain variable region according to any one of SEQ ID NO: 58-81.

2. The antibody or variant thereof of claim 1, wherein said LAG-3 binding variable domain binds to LAG-3 extracellular domain 1, 2, 3 or 4.

3. The antibody or variant thereof of claim 1, wherein the variable domain that binds an extracellular part of PD-1 is defined as a variable domain that when in a bivalent monospecific antibody that comprises two of said variable domains that bind PD-1, inhibits PD-1/PD-L1 mediated inhibition in a Jurkat cell in a range of 20-150% when compared to the inhibition obtained with the antibody Nivolumab on a Jurkat cell.

4. The antibody or variant thereof of claim 1, wherein said variable domain that binds PD-1 comprises a heavy chain variable region with a CDR1, CDR2 and CDR3 region that comprises the amino acid sequence of the CDR1, CDR2 and CDR3 of a heavy chain variable region according to any one of SEQ ID NO: 47-57.

5. The antibody or variant thereof of claim 1, wherein the variable domain that can bind to an extra cellular part of PD-1 and comprises a heavy chain variable region that comprises the amino acid sequence of the heavy chain variable region according to any one of SEQ ID NO: 47-57, having at most 15 amino acid insertions, deletions, substitutions or a combination thereof.

6. The antibody or variant thereof of claim 1, comprises a variable domain that can bind to an extra cellular part of LAG3 and comprises a heavy chain variable region that comprises the amino acid sequence of the heavy chain variable region according to any one of SEQ ID NO: 58-81, having at most 15 amino acid insertions, deletions, substitutions or a combination thereof.

7. The antibody or variant thereof of claim 1, wherein the antibody comprises: a variable domain that can bind to an extra cellular part of LAG-3 which comprises a heavy chain variable region that comprises the amino acid sequence of the heavy chain variable region according to any one of SEQ ID NO: 73, 74, 78, 79, 81, having at most 15 amino acid insertions, deletions, substitutions or a combination thereof; and a variable domain that can bind to an extra cellular part of PD-1 which comprises a heavy chain variable region that comprises the amino acid sequence of the heavy chain variable region according to SEQ ID NO: 47, having at most 15, amino acid insertions, deletions, substitutions or a combination thereof.

8. The antibody or variant thereof of claim 1 which comprises a light chain variable region having the CDR1, CDR2 and CDR3 sequences of the light chain variable region according to SEQ ID NO: 30, or CDR1, CDR2 and CDR3 sequences that deviate in no more than three amino acids from the light chain variable region CDR1 and CDR2 and CDR3 sequences of the light chain variable region according to SEQ ID NO: 30.

9. The antibody or variant thereof of claim 1 which comprises a light chain variable region having a sequence that is at least 80% identical to the amino acid sequence according to SEQ ID NO: 30.

10. A composition or kit of parts comprising at least one antibody or variant thereof according to claim 1.

11. A pharmaceutical composition comprising at least one antibody or variant thereof of claim 1 and a pharmaceutically acceptable carrier, diluent or excipient.

12. A nucleic acid molecule with a length of at least 15 nucleotides encoding at least one CDR region or encoding at least a heavy chain variable region of the variable domain that can bind to an extracellular part of LAG3 as defined in claim 1.

13. The nucleic acid molecule according to claim 12, encoding a heavy chain variable region according to any one of SEQ ID NO: 58-81.

14. A vector comprising a nucleic acid molecule or functional equivalent thereof according to claim 12.

15. An isolated or recombinant cell, or a non-human animal, comprising a nucleic acid molecule according to claim 12.

16. A method for treating a cancer or an infection with a pathogen comprising administering to a subject in need thereof a therapeutically effective amount of an antibody or variant of claim 1.

17. A method for producing an antibody or variant thereof of claim 1 from a single cell, wherein said antibody or variant thereof comprises two CH3 domains that are capable of forming an interface, said method comprising providing: a cell having a) a first nucleic acid molecule encoding a IgG heavy chain that specifically recognizes an extracellular part of a PD-1, and that contains a 1st CH3 domain, and b) a second nucleic acid sequence encoding a IgG heavy chain that specifically recognizes an extracellular part of LAG-3, and that contains a 2nd CH3 domain, wherein said nucleic acid sequences encode residues for preferential pairing of said 1st and 2nd CH3 domains, said method further comprising the step of culturing said cell and allowing for expression of said nucleic acid sequences and harvesting said antibody or variant thereof from the culture.

18. The method according to claim 17, wherein said cell has a third nucleic acid sequence encoding a common light chain.

19. The method according to claim 17, wherein said first nucleic acid encodes said first CH3 domain comprising the amino acid substitutions L351K and T366K (numbering according to the EU numbering) and wherein said second nucleic acid encodes said second CH3 domain comprising the amino acid substitutions L351D and L368E (numbering according to the EU numbering), said method further comprising the step of culturing said cell and allowing for expression of said nucleic acid sequences and harvesting said antibody or variant thereof from the culture.

20. The antibody or variant thereof of claim 1, wherein the antibody comprises: a variable domain that can bind to an extracellular part of LAG-3 which comprises a heavy chain variable region that comprises the amino acid sequence of the heavy chain variable region according to any one of SEQ ID NO: 73, 74, 78, 79, 81, having at most 15 amino acid insertions, deletions, substitutions or a combination thereof, and a variable domain that can bind to an extracellular part of PD-1 which comprises a heavy chain variable region that comprises the amino acid sequence of the heavy chain variable region according to SEQ ID NO: 56, having at most 15 amino acid insertions, deletions, substitutions or a combination thereof.

21. The antibody or variant thereof of claim 1, wherein the antibody comprises: a variable domain that can bind to an extracellular part of LAG-3 which comprises a heavy chain variable region that comprises the amino acid sequence of the heavy chain variable region according to any one of SEQ ID NO: 73, 74, 78, 79, 81, having at most 15 amino acid insertions, deletions, substitutions or a combination thereof, and a variable domain that can bind to an extracellular part of PD-1 which comprises a heavy chain variable region that comprises the amino acid sequence of the heavy chain variable region according to SEQ ID NO: 51, having at most 15, amino acid insertions, deletions, substitutions or a combination thereof.

22. The method according to claim 18, wherein the common light chain is the rearranged germline human kappa light chain IgVκ1-39*01/IGJκ1*01.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1. Common light chain used in mono- and bispecific IgG. FIG. 1A: Common light chain amino acid sequence. FIG. 1: Common light chain variable domain DNA sequence and translation (IGKV1-39/jk1). FIG. 1C: Common light chain constant region DNA sequence and translation. FIG. 1D: IGKV1-39/jk5 common light chain variable domain translation. FIG. 1E: V-region IGKV1-39A

(2) FIG. 2. IgG heavy chains for the generation of bispecific molecules. FIG. 2A: VH gene. FIG. 2B: CH1 region. FIG. 2C: hinge region. FIG. 2D: CH2 region. FIG. 2E: CH2 region containing L235G and G236R silencing substitutions. FIG. 2F: CH3 domain containing substitutions L351K and T366K (KK). FIG. 2G; CH3 domain containing substitutions L351D and L368E (DE)

(3) FIG. 2A VH: dependent on the MF (target): FIGS. 3A-3F.

(4) FIG. 2B CH1:

(5) FIG. 2C Hinge:

(6) FIG. 2D CH2:

(7) FIG. 2E CH2 containing L235G and G236R silencing substitutions:

(8) FIG. 2F CH3: KK of DEKK

(9) FIG. 2G CH3: DE of DEKK

(10) FIGS. 3A-3F. Amino acid sequences of heavy chain variable regions:

(11) FIG. 3A and 3B heavy chain variable regions of PD-1 specific clones

(12) FIG. 3C-3F heavy chain variable regions of LAG-3 specific clones

(13) The notation MF refers to a fab containing a heavy chain variable region as depicted and a common light chain. The amino acid sequence of the light chain is indicated in FIG. 1A. The underlined sequences indicate per amino acid sequence respectively the CDR1, the CDR2 and the CDR3 region.

(14) FIG. 4. Vector map and features of pIRES-Neo3 (MV1363).

(15) FIG. 5. Vector map and features of pVAX1.

(16) FIG. 6. Vector map and features of the phagemid vector MV1473 used to generate ‘immune’ phage display libraries.

(17) FIG. 7. Vector map and features of the IgG expression vector MV1452, that was used for expression of the PD-1 and PD-L1 specific Fab arms in the KK-variant heavy chain for bispecific IgG generation.

(18) FIG. 8. Amino acid sequence of the VH gene that is tetanus toxin specific when combined with the common light chain as MF1337, and that is present in the DE-variant heavy chain that was used to generate PD-L1×TT and PD-1×TT bispecific IgG molecules. The underlined sequences indicate per amino acid sequence respectively the CDR1, the CDR2 and the CDR3 region.

(19) FIG. 9. Vector map and features of the IgG expression vector MV1377, that was used for expression of the TT specific Fab arm MF1337 in the DE-variant heavy chain for bispecific IgG generation.

(20) FIG. 10. PD-1/PD-L1 blocking assay

(21) Assessment of the capacity of the anti-PD-1 antibody panel to block the interaction of PD-L1 to coated PD-1 at a concentration of 10 μg/ml bispecific IgG. Data are normalized to data obtained with the bivalent benchmark PD-L1 antibody MPDL3280A at a concentration of 10 μg/ml (100% blocking). A representative example is shown of the PD-1 panel. Maximum binding (normalized to 0% blocking) was established by incubation with a non-PD-1/PD-L1 specific human isotype antibody. All PD-1 variable domains comprising MF sequences depicted in FIGS. 3A-3F and not represented here block the PD-1/PD-L1 interaction >70%.

(22) FIG. 11

(23) LAG-3 functional activity of a panel of antibodies in a dose titration in the LAG-3 blockade reporter assay. The relation between domain mapping and LAG-3 blocking activity is shown in the LAG-3 is schematically drawn in the right panel.

(24) FIG. 12

(25) Stimulation index of IL-2 production of LAG-3×PD-1 antibodies in comparison to their parental bivalent LAG-3 antibody. Each IL-2 value is compared to the negative control antibody (not shown) to determine the SI.

(26) FIG. 13

(27) SEB-stimulation of IL-2 production in healthy donor blood cells is enhanced by anti-LAG-3×PD-1 bispecific antibodies in comparison to the parental PD-1 bivalent antibody and 25F7 LAG-3 reference antibody.

(28) FIG. 14

(29) Comparator anti-LAG-3 antibodies. PG1337P300 is a control antibody that is not expected to bind to the cells and binds tetanus toxoid.

(30) Left hand panel shows binding of the indicated antibodies to 293FF-LAG-3 cells that express LAG-3 on the cell membrane. Right hand panel shows activated human T-cells. Binding of antibody to the cells was detected with PE-labelled anti-IgG F(ab′)2. 25F7*, determined affinity ˜0.2 nM.

(31) FIG. 15

(32) FACS-based LAG-3 panel characterization. Nineteen LAG-3 specific antibodies were expressed in monovalent (PB LAG-3×TT) and bivalent (PG) format. Antibody binding was tested on activated T cells and 293FF-LAG-3 stable cell lines. The two panels show an example of MF7116 and MF7431 (25F7*) mono- and bivalent binding on 293FF-LAG-3 cells. Monovalent binding was determined with a bispecific antibody (PB) where one arm has a VH (indicated by the letters MF) of the indicated LAG-3 antibody. The other arm of the antibody has a VH specific for tetanus toxoid (MF1337). Differences in binding were observed between some bivalent/monovalent formats of LAG-3 panel, but not of 25F7*.

(33) FIG. 16

(34) LAG-3×PD-1 reporter assay validation: bispecific antibodies show activity in LAG-3×PD-1 reporter assay.

(35) FIG. 17

(36) PD-1+LAG-3 reporter assay screening. Panel A, LAG-3×PD-1 with functional LAG-3 and PD-1 arms. Panel B, LAG-3×PD-1 with non-functional PD-1 arm with MF5374. Panel C, LAG-3×PD-1 with non-functional LAG-3 arms with MF7118 and MF7167. * indicates surrogate arms.

(37) FIG. 18

(38) Summary of the results reporter assay screening set out in FIG. 18. The four digit number immediately following the letters MG indicates the MF number of the heavy chain variable region of the variable domain of the bispecific antibody. For example, the bispecific antibody with the result 52,22 in the upper left hand corner of the table has one arm with the MF6930 and one arm with MF7518. Values are area under the curve (AUC) as % of control.

(39) FIG. 19

(40) SEB assay screening: example IL-2 production. Panel A, LAG-3×PD-1 with functional PD-1 and LAG-3 arms. Panel B, LAG-3×PD-1 with non-functional PD-1 arm with VH MF5374. Panel C, LAG-3×PD-1 with non-functional LAG-3 arms with VH MF7118 and MF7167. * indicates a surrogate antibody.

(41) FIGS. 20A-20C

(42) Screening results reporter and SEB assays. Arms ranked on (high to low) functionality as bivalent antibodies in reporter assay. Top in each of the columns a LAG-3 arm, Left in each of the rows a PD-1 arm. As indicated for FIG. 19, the four digit number immediately following the letters MG indicates the MF number of the heavy chain variable region of the variable domain of the bispecific antibody. Values are AUC as % of control: White=Top 15%: Grey=Middle 43%: Black=Lowest 43%.

(43) FIG. 21

(44) Ranking LAG-3×PD-1; PD-1 arms. Matrix files were used to define the Top 15% of bispecific antibodies based on AUC as compared to positive control in: 1) Reporter assay; 2) SEB screening donor 1 (IL-2 data); 3) SEB screening donor 2 (IL-2 data). Next, it was scored how many bispecific antibodies carrying a specific PD-1 Fab arm were present in this top 15%. Clones with PD-1 arms having a variable domain with a VH of MF6974 or a VH of MF6076 performed best in combination with most of the LAG-3 arms in the reporter and SEB assays. Color/Grey coding, darker indicates higher rank.

(45) FIG. 22

(46) Ranking LAG-3×PD-1; LAG-3 arms. Matrix file was used to define the Top 15% (left) and top 25% (right) of Bispecifics based on AUC as compared to positive control in: 1) Reporter assay; 2) SEB screening donor 1 based on IL-2 data; 3) SEB screening donor 2 based on IL-2 data. Next, it was scored how many bispecific antibodies carrying a specific LAG-3 Fab arm were present in this top 15%. Fab arms with same score in Top 15% were further ranked by using Top 25% scores. Color/Grey coding, darker indicates higher rank.

(47) FIG. 23

(48) Ranking of PD1 arms. PD-1 arms (left hand column) based on ranking, sequence diversity and binding affinity.

(49) FIG. 24

(50) Ranking of LAG-3 arms. LAG-3 arms (left hand column) based on ranking, sequence diversity and binding affinity.

(51) FIG. 25.

(52) Effect of test antibodies on IFN-γ production in allogeneic mMLR.Mo-DCs were prepared from CD14+ monocytes cultured for 7 days. Immature DCs were used on day 7 and mature DCs were generated by culturing for a further 3 days in maturation medium before being cultured together with T cells isolated by negative selection and test antibody for 4 days (mMLR). IFN-y was measured in culture supernatants by ELISA. Data are normalized to vehicle control. Four separate MLRs were performed.

EXAMPLES

(53) As used herein “MFXXXX” wherein X is independently a numeral 0-9, refers to a Fab comprising a variable domain wherein the VH has the amino acid sequence identified by the 4 digits. Unless otherwise indicated the light chain variable region of the variable domain typically has a sequence of FIG. 1A, typically 1B. “MFXXXX VH” refers to the amino acid sequence of the VH identified by the 4 digits. The MF further comprises a constant region of a light chain and a constant region of a heavy chain that normally interacts with a constant region of a light chain. PG refers to a monospecific antibody comprising identical heavy and light chains. PB refers to a bispecific antibody with two different heavy chains. The variable region of the heavy chains differs and typically also the CH3 region, wherein one of the heavy chains has a KK mutation of its CH3 domain and the other has the complementing DE mutation of its CH3 domain (see for reference PCT/NL2013/050294 (published as WO2013/157954).

Example: 1

(54) Generation of Materials for Selection and Screening

(55) Culturing of Cell Lines

(56) Freestyle 293F cells (cat. no. p/n51-0029) were obtained from Invitrogen and routinely maintained in 293 FreeStyle medium. HEK293T (cat. no. ATCC-CRL-11268) cells were purchased from ATCC and routinely maintained in DMEM/F12 (Gibco) supplemented with L-Glutamine (Gibco) and FBS (Lonza), and CHO-S (cat. no. 11619-012) cell lines were purchased from Gibco and routinely maintained in Freestyle CHO expression medium (Invitrogen) supplemented with L-glutamine.

(57) Generation of PD-1 and LAG-3 Expression Vectors for Immunization, and for Generation of Stable Cell Lines and Transient Transfections

(58) Full length cDNA of each target including unique restriction sites for cloning and kozak consensus sequence for efficient translation was either synthetized, or obtained via PCR amplification on a commercially available expression construct, containing the target cDNA, with specific primers that introduced unique restriction sites for cloning and kozak consensus sequence for efficient translation. The cDNA of each target was cloned into a eukaryotic expression construct such as pIRES-Neo3 (Clontech; FIG. 4) or pVAX1 (Thermo Fisher Scientific; FIG. 5) via NheI/EcoRI, resulting in pIRES-Neo3_[TARGET_NAME] and pVAX1_[TARGET_NAME], respectively. The insert sequences were verified by comparison with NCBI Reference amino acid sequences. The pIRES-Neo3 constructs were used for generation of stable cell lines and transient transfections. The pVAX1 constructs were used for immunization purposes. See TABLE 1 for an overview of the names of the resulting constructs.

(59) Amino acid sequence full length huPD-1 insert (both in pIRES-Neo3 and pVAX1) for expression on the cell surface (Identical to GenBank: NP_005009.2):

(60) TABLE-US-00001 (SEQ ID NO: 1) MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEG DNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCR FRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAE LRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVL AVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTP EPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDG HCSWPL Of which: (SEQ ID NO: 125) MQIPQAPWPVVWAVLQLGWR: signal peptide. (SEQ ID NO: 126) PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNW YRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRAR RNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPR PAGQFQTLV: ECD of huPD-1. (SEQ ID NO: 127) VGVVGGLLGSLVLLVWVLAVI: Predicted TM region. (SEQ ID NO: 128) CSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPP VPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCS WPL: Intracellular tail.

(61) Amino acid sequence full length macaque (Macaca fascicularis) PD-1 insert (both in pIRES-Neo3 and pVAX1) for expression on the cell surface (Identical to GenBank: ABR15751.1):

(62) TABLE-US-00002 (SEQ ID NO: 2) MQIPQAPWPVVWAVLQLGWRPGWFLESPDRPWNAPTFSPALLLVTEG DNATFTCSFSNASESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCR FRVTRLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAE LRVTERRAEVPTAHPSPSPRPAGQFQALVVGVVGGLLGSLVLLVWVL AVICSRAAQGTIEARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTP EPPAPCVPEQTEYATIVFPSGLGTSSPARRGSADGPRSPRPLRPEDG HCSWPL Of which: (SEQ ID NO: 125) MQIPQAPWPVVWAVLQLGWR: signal peptide. (SEQ ID NO: 129) PGWFLESPDRPWNAPTFSPALLLVTEGDNATFTCSFSNASESFVLNW YRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTRLPNGRDFHMSVVRAR RNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPR PAGQFQALV: ECD of maPD-1. (SEQ ID NO: 127) VGVVGGLLGSLVLLVWVLAVI: Predicted TM region. (SEQ ID NO: 130) CSRAAQGTIEARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPP APCVPEQTEYATIVFPSGLGTSSPARRGSADGPRSPRPLRPEDGHCS WPL: Intracellular tail.

(63) TABLE-US-00003 (SEQ ID NO: 3) MWEAQFLGLLFLQPLWVAPVKPLQPGAEVPVVWAQEGAPAQLPCSPT IPLQDLSLLRRAGYTWQHQPDSGPPAAAPGHPLAPGPHPAAPSSWGP RPRRYTVLSVGPGGLRSGRLPLQPRVQLDERGRQRGDFSLWLRPARR ADAGEYRAAVHLRDRALSCRLRLRLGQASMTASPPGSLRASDWVILN CSFSRPDRPASVHWFRNRGQGRVPVRESPHHHLAESFLFLPQVSPMD SGPWGCILTYRDGFNVSIMYNLTVLGLEPPTPLTVYAGAGSRVGLPC RLPAGVGTRSFLTAKWTPPGGGPDLLVTGDNGDFTLRLEDVSQAQAG TYTCHIHLQEQQLNATVTLAIITVTPKSFGSPGSLGKLLCEVTPVSG QERFVWSSLDTPSQRSFSGPWLEAQEAQLLSQPWQCQLYQGERLLGA AVYFTELSSPGAQRSGRAPGALPAGHLLLFLILGVLSLLLLVTGAFG FHLWRRQWRPRRFSALEQGIHPPQAQSKIEELEQEPEPEPEPEPEPE PEPEPEQL Of which: (SEQ ID NO: 131) MWEAQFLGLLFLQPLWVAPVKP:  signal peptide. (SEQ ID NO: 132) LQPGAEVPVVWAQEGAPAQLPCSPTIPLQDLSLLRRAGVTWQHQPDS GPPAAAPGHPLAPGPHPAAPSSWGPRPRRYTVLSVGPGGLRSGRLPL QPRVQLDERGRQRGDFSLWLRPARRADAGEYRAAVHLRDRALSCRLR LRLGQASMTASPPGSLRASDWVILNCSFSRPDRPASVHWFRNRGQGR VPVRESPHHHLAESFLFLPQVSPMDSGPWGCILTYRDGFNVSIMYNL TVLGLEPPTPLTVYAGAGSRVGLPCRLPAGVGTRSFLTAKWTPPGGG PDLLVTGDNGDFTLRLEDVSQAQAGTYTCHIHLQEQQLNATVTLAII TVTPKSFGSPGSLGKLLCEVTPVSGQERFVWSSLDTPSQRSFSGPWL EAQEAQLLSQPWQCQLYQGERLLGAAVYFTELSSTGAQRSGRAPGAL PAGHL:  ECD. (SEQ ID NO: 133) LLFLILGVLSLLLLVTGAFGF:  Predicted TM region. (SEQ ID NO: 134) HLWRRQWRPRRFSALEQGIHPPQAQSKIEELEQEPEPEPEPEPEPEP EPEPEQL: Intracellular tail.

(64) Amino acid sequence full length rat (Rattus norvegicus) LAG-3 insert (both in pIRES-Neo3 and pVAX1) for expression on the cell surface (Identical to GenBank: NP_997678.2):

(65) TABLE-US-00004 (SEQ ID NO: 4) MRQDLFLDLLLLQLLWEAPVVSSGPGKELSVVWAQEGAPVHLPCSLE FPHLDPNFLRRGWVTWQHRPDSDQPASIPALDLLQGMPSTRRHPPHR YTVLSVAPGGLRSGRQPLLSHVQLEKRGPQRGDFSLWLRPATRKDAG EYHAFVRLPDRDFSCSLRLRVGQASMIASPPGTLKPSDWVILNCSFS RPDRPVSVHWFQGQSRVPVHNSPRHYLAESFLLLPQVSPLDSGTWGC VLTYRDGFNVSITYNLKVQGLEPVAPLTVYAAEGSRVELPCHLPPVV GTPSLLIAKWTPPGGGPELPVTGKSGNFTLQLENVGRAQAGTYTCSI HLQGRQLSAAVTLAVITVTPKSFGLPGSPQKLLCEVVPASGEGRFVW RPLSDLSRSSLGPVLELQEAKLLAEQWQCQLYEGQKLLGATVYTAES SSGAWSAKRISGDLKGGHLFLSLILGALALFLLVTGAFGFHLWRRQL LRRRFSALEHGIRPPPVQSKIEELEREPETEMEPETEPDPEPQPEPE LEPESRQL Of which: (SEQ ID NO: 135) MRQDLFLDLLLLQLLWEAPVVSS:  signal peptide. (SEQ ID NO: 136) GPGKELSVVWAQEGAPVHLPCSLEFPHLDPNFLRRGWVTWQHRPDSD QPASIPALDLLQGMPSTRRHPPHRYTVLSVAPGGLRSGRQPLLSHVQ LEKRGPQRGDFSLWLRPATRKDAGEYHAFVRLPDRDFSCSLRLRVGQ ASMIASPPGTLKPSDWVILNCSFSRPDRPVSVHWFQGQSRVPVHNSP RHYLAESFLLLPQVSPLDSGTWGCVLTYRDGFNVSITYNLKVQGLEP VAPLTVYAAEGSRVELPCHLPPVVGTPSLLIAKWTPPGGGPELPVTG KSGNFTLQLENVGRAQAGTYTCSIHLQGRQLSAAVTLAVITVTPKSF GLPGSPQKLLCEVVPASGEGRFVWRPLSDLSRSSLGPVLELQEAKLL AEQWQCQLYEGQKLLGATVYTAESSSGAWSAKRISGDLKGGHL: ECD. (SEQ ID NO: 137) FLSLILGALALFLLVTGAFGF:  Predicted TM region. (SEQ ID NO: 138) HLWRRQLLRRRFSALEHGIRPPPVQSKIEELEREPETEMEPETEPDP EPQPEPELEPESRQL:  Intracellular tail.

(66) Amino acid sequence full length macaque (Macaca mulatta) LAG-3 insert (both in pIRES-Neo3 and pVAX1) for expression on the cell surface (Identical to GenBank: Macaca mulatta):

(67) TABLE-US-00005 (SEQ ID NO: 5) MWEAQFLGLLFLQPLWVAPVKPPQPGAEISVVWAQEGAPAQLPCSPT IPLQDLSLLRRAGVTWQHQPDSGPPAPAPGHPPAPGHRPAAPYSWGP RPRRYTVLSVGPGGLRSGRLPLQPRVQLDERGRQRGDFSLWLRPARR ADAGEYRATVHLRDRALSCRLRLRVGQASMTASPPGSLRTSDWVILN CSFSRPDRPASVHWFRSRGQGRVPVQGSPHHHLAESFLFLPHVGPMD SGLWGCILTYRDGFNVSIMYNLTVLGLEPATPLTVYAGAGSRVELPC RLPPAVGTQSFLTAKWAPPGGGPDLLVAGDNGDFTLRLEDVSQAQAG TYICHIRLQGQQLNATVTLAIITVTPKSFGSPGSLGKLLCEVTPASG QEHFVWSPLNTPSQRSFSGPWLEAQEAQLLSQPWQCQLHQGETLLGA AVYFTELSSPGAQRSGRAPGALRAGHLPLFLILGVLFLLLLVTGAFG FHLWRRQWRPRRFSALEQGIHPPQAQSKIEELEQEPELEPEPELERE LGPEPEPGPEPEPEQL Of which: (SEQ ID NO: 131) MWEAQFLGLLFLQPLWVAPVKP:  signal peptide. (SEQ ID NO: 139) PQPGAEISVVWAQEGAPAQLPCSPTIPLQDLSLLRRAGVTWQHQPDS GPPAPAPGHPPAPGHRPAAPYSWGPRPRRYTVLSVGPGGLRSGRLPL QPRVQLDERGRQRGDFSLWLRPARRADAGEYRATVHLRDRALSCRLR LRVGQASMTASPPGSLRTSDWVILNCSFSRPDRPASVHWFRSRGQGR VPVQGSPHHHLAESFLFLPHVGPMDSGLWGCILTYRDGFNVSIMYNL TVLGLEPATPLTVYAGAGSRVELPCRLPPAVGTQSFLTAKWAPPGGG PDLLVAGDNGDFTLRLEDVSQAQAGTYICHIRLQGQQLNATVTLAII TVTPKSFGSPGSLGKLLCEVTPASGQEHFVWSPLNTPSQRSFSGPWL EAQEAQLLSQPWQCQLHQGETLLGAAVYFTELSSPGAQRSGRAPGAL RAGHL: ECD. (SEQ ID NO: 140) PLFLILGVLFLLLLVTGAFGF:  Predicted TM region. (SEQ ID NO: 141) HLWRRQWRPRRFSALEQGIHPPQAQSKIEELEQEPELEPEPELEREL GPEPEPGPEPEPEQL:  Intracellular tail.
Generation of Stable Cell Lines Expressing PD-1 or LAG-3

(68) pIRES-Neo3_[TARGET_NAME] expression constructs (TABLE 1) were used to generate CHO-S or Freestyle 293F clones stably expressing the respective proteins. Constructs were transiently transfected in CHO-S and Freestyle 293F cells using lipofectamine transfection, and screened by FACS using antibodies reacting with the respective proteins. After confirmation of expression, transiently transfected cells were seeded in limiting dilution and cultured under selection pressure relevant for the used expression construct to obtain stable cell clones. After 2-3 weeks of selection, clones were screened by FACS. The selected clones were expanded by serial passage, retested in FACS and frozen to −150° C. The names of clones that stably express the heterologous proteins are CHO-S_[TARGET_NAME] cells or Freestyle 293F_[TARGET_NAME] cells. See TABLE 1 for an overview of the constructs used to generate the stable cell lines and their resulting name.

Example 2

(69) Immunization, Selection and Screening

(70) Mice Used for Immunizations

(71) For generation of human antibodies binding to huPD-1 and huLAG-3, mice transgenic for the human VK1-39 light chain (common light chain mice, see WO2009/157771) and for a human heavy chain (HG) minilocus (comprising a selection of human V gene segments, all human Ds and all human Js) were immunized. These mice are referred to as ‘MeMo®’ mice. Mice were immunized with either recombinant protein antigen, or DNA encoding the protein antigen as briefly described below.

(72) Protein Immunizations

(73) ‘MeMo®’ mice were immunized by subcutaneous injections with recombinant protein and Gerbu adjuvant MM (Gerbu Biotechnik; cat. no. 3001). Recombinant huPD-1-Fc (R&D; cat. no. 1086-PD) and huLAG-3-His (Abcam; cat. no. Ab184729) were used for immunizations. Mice were immunized with 40 μg recombinant protein in PBS mixed with 40 μl of adjuvant in a total volume of 100 μl. Subsequently mice were boosted on day 14 and 28 with 20 μg of recombinant protein in PBS together with 20 μl of adjuvant in a total volume of 50 μl. Mouse serum was collected at day 35 to determine serum titers. Mice with low serum reactivity against the human and/or macaque target received additional cycles of booster immunizations with recombinant human or macaque protein antigen and serum analyses. Each cycle consisted of two weekly immunizations using to 20 μg of recombinant protein in 50 μl PBS followed one week later by serum collection for titer analysis. Mice showing high serum titers against the human and macaque target received a final boost immunization consisting of daily injections with 20 μg of recombinant protein in 50 μl PBS on three consecutive days. One day after the final injection mouse lymphoid tissue was collected.

(74) DNA Immunizations

(75) MeMo®’ mice were immunized by DNA tattooing using a micropigmentation device. DNA tattoo immunizations were performed with 20 μg plasmid DNA encoding the target antigen (pVAX1_[TARGET_NAME], TABLE 1). Mice were immunized with DNA encoding the human target only (PD-1 and LAG-3) or by alternating immunizations with DNA encoding the human and rat (LAG-3) target to obtain species cross-reactive antibodies. Mice were immunized at day 0, 3, 6, 14, 17, 28 and 31. Mouse serum was collected at day 35 to determine serum titers. Mice with low serum reactivity against the human and/or macaque target received additional cycles of booster immunizations with human DNA antigen, and serum analyses. Each cycle consisted of two weekly DNA immunizations followed one week later by serum collection for titer analysis. Mice showing strong serum reactivity against cells expressing the human and macaque target received a final boost immunization followed after 3 days by collection of lymphoid tissue.

(76) Determination of Serum Titers

(77) Serum titers were determined by FACS analysis using cell lines expressing the human and macaque target antigens.

(78) Generation of Synthetic Phage Fab Libraries

(79) Synthetic libraries were constructed based on a repertoire of germline human VH genes that were selected for frequent use in natural repertoires and canonical sequence diversity. Synthetic HCDR3 regions were added to these VH genes using PCR. This was done using forward primers that anneal to framework 1 of the VH genes and include a SfI restriction site for cloning. Reverse primers included sequences to anneal to framework 3 of the VH genes, followed by randomized sequences to encode HCDR3 diversity and a framework 4 encoding sequence also containing a BstEII and XhoI restriction site for cloning. Synthetic CDR3 regions were either completely random or encoded a more restricted diversity based on the frequency of use of amino acid residues at certain positions within the HCDR3. PCR products encoding the VH genes were cloned into phage display vectors in fusion with phage M13 gene 3 protein using aforementioned restriction enzymes and also containing a common light chain encoding gene. Large scale ligation and transformation of E′coli TG1 resulted in large libraries of synthetic Fab fragments displayed on phage which were used for panning on antigens or cells to identify antigen-specific Fab fragments.

(80) Generation of ‘Immune’ Phage Fab Libraries by RT-PCR from Tissues of Immunized Mice

(81) Spleen and draining lymph nodes were removed from mice for which a significant humoral response was observed against the respective target proteins. Single cell suspensions were generated from both spleen and inguinal lymph nodes and subsequently these tissues were lysed in Trizol LS Reagent (Thermo Scientific c #10296028) and stored at −80° C. until use.

(82) From successfully immunized mice, the inguinal lymph nodes were used for the construction of ‘immune’ phage antibody repertoires. RNA was extracted from the single cell suspensions of the lymphoid tissue. 1 μg of total RNA was used in a RT reaction using an IgG-CH1 specific primer. The resulting cDNA was then used to amplify the polyclonal pool of VH-encoding cDNA using in-house adapted VH-specific primers essentially as described in Marks et al. (J Mol Biol. 1991 Dec. 5; 222(3):581-97). The resulting PCR product was then cloned in a phagemid vector (FIG. 6) for the display of Fab fragments on phage, as described in de Haard et al. (J Biol Chem. 1999 Jun. 25; 274(26):18218-30) with the exception that the light chain (FIGS. 1A and 1B) was the same for every antibody and was encoded by the vector. After ligation, the phagemids were used to transform E. coli TG1 bacteria and transformed bacteria were plated onto LB-agar plates containing ampicillin and glucose. All phage libraries contained >4×10.sup.5 transformants and had an insert frequency of >90%. Bacteria were harvested after overnight growth and used to prepare phage according to established protocols (de Haard et al., J Biol Chem. 1999 Jun. 25; 274(26):18218-30).

(83) Selection of Phage Carrying Fab Fragments Specifically Binding to Human Target Protein from Synthetic and ‘Immune’ Phage Fab Libraries Using Recombinant Proteins

(84) The phage Fab libraries that were generated were used to select target specific Fabs using phage display on directly coated recombinant proteins. For PD-1, huPD-1-Fc (R&D; cat. no. 1086-PD) and huPD-1 biotin (BPS bioscience; cat. no. 71109) were used. For LAG-3, huLAG-3-Fc (R&D; cat. no. 2319-L3), huLAG-3-Fc (Enzo; cat. no. ALX-522-078), huLAG-3-His (Abcam; cat. no. Ab184729) and ratLAG-3 His (SinoBiological; cat. no. 80367-R08H) were used.

(85) For selections with non-biotinylated recombinant protein (‘panning selections’), proteins were coated onto the wells of a MAXISORP™ ELISA plate. The MAXISORP™ ELISA plates were blocked with 4% dried skimmed milk (Marvel) in PBS. Phage Fab libraries were also blocked with 4% Marvel and, when Fe tagged recombinant protein was used, also with excess of human IgG to deplete for Fc region binders prior to the addition of the phage library to the coated antigen.

(86) Incubation of the phage library with the coated protein was performed for 1.5 hrs at room temperature under shaking conditions. Plates or tubes were then washed fifteen times with 0.05% Tween-20 in PBS followed by 5 times washing with PBS. Bound phage were eluted for 20 minutes using trypsin, after which trypsin was neutralized with AEBSF trypsin inhibitor (Sigma).

(87) For selections with biotinylated protein (‘in-solution selections’), neutravidin was coated onto the well of a MAXISORP™ ELISA plate. The MAXISORP™ ELISA plates were blocked with 1% casein in PBS. In parallel, biotinylated protein and phage Fab libraries were blocked for 30 minutes in 0.5% casein in PBS, containing an excess of human IgG, in separate Eppendorf tubes. Thereafter, the blocked phage and biotinylated protein were mixed and incubated for 2 hours at room temperature. The mixture was thereafter added to the neutravidin coated wells for 20 minutes to capture the phage Fab particles that were bound to biotinylated protein. Plates were then washed fifteen times with 0.05% Tween-20 in PBS followed by 5 times washing with PBS. Bound phage were eluted for 20 minutes using trypsin, after which trypsin was neutralized with AEBSF trypsin inhibitor (Sigma).

(88) The eluates of both selection strategies (‘panning and in-solution’) were added to E. coli TG-1 and incubated at 37° C. for phage infection. Subsequently infected bacteria were plated on agar plates containing Ampicillin and glucose, and incubated at 37° C. overnight. Single clones from the selection outputs were screened for target binding in ELISA or FACS depending on the target.

(89) For selections with synthetic phage Fab libraries, a second round selection was performed after rescue of the first round selection output using the same protocol as outlined above for the first round selection. The same selection antigen that was used in the first round was also used in the second round, with exception of first round raLAG-3-His selections that were followed by a second round selection with huLAG-3-His.

(90) Selection of Phage Carrying Fab Fragments Specifically Binding to Human Target from ‘Immune’ Phage Fab Libraries Using Cells Stably Expressing the Target Protein

(91) Phage Fab libraries that were generated from target immunized mice were selected using phage display on cells expressing the respective target. The stable cell lines expressing PD-1 or LAG-3 (Table 1) were used for 1.sup.st round selections. Cells were blocked with 10% FBS in PBS. After blocking, the rescued phage were incubated with blocked cells. Cells plus phage were incubated for 1 hr at 4° C. Washing the cells (5 times) was performed using 1 ml of 10% FBS in PBS. Bound phage were eluted using trypsin for 20 minutes, after which trypsin was neutralized with AEBSF trypsin inhibitor (Sigma). The eluate was added to E. coli TG-1 and incubated at 37° C. for phage infection. Subsequently, phage-infected bacteria were plated on agar plates containing ampicillin and glucose, and incubated at 37° C. overnight.

(92) Screening for Target Specific Fab Clones in ELISA

(93) Of single clones, soluble Fab or phage were prepared (J Mol Biol. 1991 Dec. 5; 222(3):581-97; J Biol Chem. 1999 Jun. 25; 274(26):18218-30). Obtained soluble Fab or phage samples were diluted (1:5 or 1:10, respectively) in 4% dried skimmed milk (Marvel) in PBS (blockbuffer) and tested for binding in ELISA to wells coated with the same antigen as was used for selection, or with huLAG-3-His (Abcam; cat. no. Ab184729) for all selection outputs performed with ratLAG-3 His (SinoBiological; cat. no. 80367-R08H).

(94) Bound Fabs were detected by staining with an anti-myc antibody (Roche; cat. no. 11667203001) diluted 1:1000 in blockbuffer, followed by a HRP-conjugated anti-mouse IgG antibody (Jackson Immunoresearch; cat. no. 715-035-150) diluted 1:5000 in blockbuffer. Bound phage were detected by staining with a HRP-conjugated monoclonal anti-M13 antibody (GE healthcare; cat. no. 27-9421-01) diluted 1:5000 in blockbuffer.

(95) After each antibody staining, wells were washed with PBS-T (PBS-0.05% v/v Tween 20). Bound secondary antibody was visualized by TMB/H.sub.2O.sub.2 staining and staining was quantified by means of OD.sub.450 nm measurement. Clones were considered to bind the target when the OD450 nm was at least three times above the background signal obtained with a negative control Fab.

(96) The VH-encoding cDNA's of all target-specific clones were sequenced. A selection of unique clones based on sequence identity and cluster analysis was then analyzed in FACS on binding to PD-L1 expressed on cells as described below for the clones obtained from the cell selection outputs.

(97) Screening for Target Specific Fab Clones in FACS

(98) Of single clones, selected on cells expressing the respective target, soluble Fab or phage were prepared as described (J Mol Biol. 1991 Dec. 5; 222(3):581-97; J Biol Chem. 1999 Jun. 25; 274(26):18218-30). Fab samples were tested for binding in FACS to cells expressing the human and macaque target (Table 1) by incubation with a mix of 1:5 diluted Fab sample with 1:1000 diluted anti-myc antibody (Gentaur; cat. no. 04-CMYC-9E10) in FACS buffer (0.5% HI-FBS in PBS). Bound Fab/anti-myc complexes were detected by incubation with an APC-conjugated goat anti-mouse IgG antibody (BD Bioscience; cat. no. 550826) diluted 1:500 in FACS buffer.

(99) Phage samples were tested for binding in FACS by diluting the phage samples 1:3 in blockbuffer and incubation with target expressing cells for 1 hour. Bound phage were detected by staining with a biotinylated anti-M13 antibody (Fitzgerald, cat. nr. 61R-M101ABTB62-FEZ, 1:125 in FACS buffer, 30 minutes on ice) and PE-labeled streptavidin (Invitrogen, cat. nr. SA1004-4; 1:400 in FACS buffer for 15 minutes on ice). After each antibody incubation, wells were washed three times with FACS buffer. Stained cells were analysed using a FACS Accuri C6 instrument (Becton and Dickinson). Clones were considered positive when the mean fluorescence intensity was at least three times above the background signal obtained with a negative control Fab.

Example 3

(100) Characterization huLAG-3 and huPD-1 Specific Fab Clones in IgG Format

(101) Recloning Human LAG-3 and PD-1 Specific Fab to IgG Format

(102) A selection of unique clones, based on CDR3 sequence and VH germline differences, that bound human and macaque target protein expressed on cells, was then re-cloned to an IgG expression plasmid such as MV1452 (FIG. 7), which contained the common light chain (FIG. 1), using Sfi1-BstEII digestion and ligation of the pool of digested cDNA's according to standardized molecular biological techniques.

(103) Expression of Bispecific IgG Containing a Human LAG-3 or Human PD-1 Specific Fab and a Tetanus Toxin Specific Fab

(104) Bispecific antibodies were generated by transient co-transfection of two plasmids encoding IgG with different VH domains, using a proprietary CH3 engineering technology to ensure efficient hetero-dimerisation and formation of bispecific antibodies. The common light chain present on both plasmids containing the heavy chain is also co-transfected in the same cell. In our co-pending applications (e.g. WO2013/157954 and WO2013/157953; incorporated herein by reference) we have disclosed methods and means for producing bispecific antibodies from a single cell, whereby means are provided that favor the formation of bispecific antibodies over the formation of monospecific antibodies. These methods can also be favorably employed in the present invention. Specifically, preferred mutations to produce essentially only bispecific full length IgG molecules are amino acid substitutions at positions 351 and 366, e.g. L351K and T366K (numbering according to EU numbering) in the first CH3 domain (the ‘KK-variant’ heavy chain) and amino acid substitutions at positions 351 and 368, e.g. L351D and L368E in the second CH3 domain (the ‘DE-variant’ heavy chain), or vice versa (FIG. 2). It was previously demonstrated in our co-pending applications that the negatively charged DE-variant heavy chain and positively charged KK-variant heavy chain preferentially pair to form heterodimers (so-called ‘DEKK’ bispecific molecules). Homodimerization of DE-variant heavy chains (DE-DE homodimers) or KK-variant heavy chains (KK-KK homodimers) hardly occurs due to strong repulsion between the charged residues in the CH3-CH3 interface between identical heavy chains.

(105) VH genes encoding the antibodies binding human LAG-3 and PD-1 described above were cloned into the MV1452 IgG expression vector encoding the positively charged CH3 domain. A tetanus toxin (TT) targeting antibody (FIG. 8) was cloned into the MV1377 IgG expression vector (FIG. 9) encoding the negatively charged CH3 domain. For expression of the LAG-3 and PD-1 antibody panel in IgG format, the entire panel was also cloned into the negatively charged CH3 domain vector to be able to produce bivalent LAG-3 or PD-1 IgG. Suspension growth-adapted 293F Freestyle cells were cultivated in T125 flasks on a shaker plateau until a density of 3.0×10.sup.6 cells/ml. Cells were seeded at a density of 0.3-0.5×10.sup.6 viable cells/ml in each well of a 24-deep well plate. The cells were transiently transfected with a mix of two plasmids encoding different antibodies, cloned into the proprietary vector system. Seven days after transfection, the cellular supernatant was harvested and filtered through a 0.22 μM filter (Sartorius). The sterile supernatant was stored at 4° C. until purification of the antibodies.

(106) Purification of Bispecific IgG

(107) Purification of IgG was performed on a small scale (<500 μg), using protein-A affinity chromatography. Small scale purifications were performed under sterile conditions in 24 well filter plates using filtration. First, the pH of the medium was adjusted to pH 8.0 and subsequently, IgG-containing supernatants were incubated with protein A Sepharose CL-4B beads (50% v/v) (Pierce) for 2 hrs at 25° C. on a shaking platform at 600 rpm. Next, the beads were harvested by filtration. Beads were washed twice with PBS pH 7.4. Bound IgG was then eluted at pH 3.0 with 0.1 M citrate buffer and the eluate was immediately neutralized using Tris pH 8.0. Buffer exchange was performed by centrifugation using multiscreen Ultracel 10 multiplates (Millipore). The samples were finally harvested in PBS pH7.4. The IgG concentration was measured using Octet. Protein samples were stored at 4° C.

(108) IgG Quantification Using Octet

(109) To determine the amount of IgG purified, the concentration of antibody was determined by means of Octet analysis using protein-A biosensors (Forte-Bio, according to the supplier's recommendations) using total human IgG (Sigma Aldrich, cat. nr. 14506) as standard.

(110) Specificity Analysis huLAG-3 and huPD-1 IgG

(111) The antibodies (bivalent LAG-3 antibodies and bispecific PD-1×TT antibodies) were tested for binding in FACS to the stable cell lines expressing the relevant human and macaque orthologs (Table 1) and the wt cells. Therefore, cells were harvested and diluted to 10.sup.6 cells/ml in FACS buffer (PBS/0.5% BSA/0.5 mM EDTA). 1-2×10.sup.5 cells were added to each well in a U-bottom 96 well plate. Cells were centrifuged for 2 minutes at 300 g at 4° C. Supernatant was discarded by inverting the plate(s). 50 μl of each IgG sample at a concentration of 10 μg/ml was added and incubated for 1H on ice. Cells were centrifuged once, supernatant was removed and cells were washed twice with 150 μl of FACS buffer. 50 μl diluted 1:400 goat anti human IgG PE (Invitrogen) was added and incubated for 30 minutes on ice in the dark. After adding FACS buffer, cells were centrifuged once, supernatant was removed and cells were washed twice with FACS buffer. Cells were analyzed on a FACSCanto Flow cytometer (Becton and Dickinson) in a HTS setting. Binding of the antibodies to cells was assessed by measuring the mean fluorescence intensity (MFI) of the stained cell population. Antibodies were considered to bind their target when the MFI was at least five-fold that of the same cell population stained with a (negative control) non-binding antibody (directed to tetanus toxoid).

(112) Binning huPD-1 Specific Fab Arms Present in the PD-1×TT Bispecific IgG on Ligand Blocking Ability

(113) huPD-1 binding clones were tested for their ability to block the interaction of PD-L1 with PD-1. Therefore PD1-Fc (R&D systems; cat. no. 1086-PD) was coated to a maxisorp plate at 1 μg/ml. Coated wells were blocked with 4% BSA in PBS. Thereafter, 0.55 μg/ml biotinylated PD-L1 (BPS bioscience; cat. no. 71105) was added in the presence or absence of IgG in the range of 0.15 to 20 μg/ml. Bound biotinylated PD-L1 was detected with HRP-conjugated streptavidin (BD bioscience: cat. no. 554066) diluted 1:2000 in block buffer. After each incubation step, the ELISA plate was washed three times with PBS-T (PBS-0.05% v/v Tween 20). Bound streptavidin was visualized by TMB/H.sub.2O.sub.2 staining and staining was quantified by means of OD.sub.450 nm measurement. Clones were considered to block the interaction of PD-1 with PD-L1 when the ELISA signal was reduced more than 70% at an IgG (PD-1×TT) concentration of 10 μg/ml, compared to a control in which a TT specific competition antibody was added. See FIG. 10 for the results obtained with a representative selection of the PD-1 antibody panel tested as PD-1×TT bispecific molecules.

(114) Affinity Ranking huLAG-3 and huPD-1 Specific Fab Arms Present in the LAG-3×TT and PD-1×TT Bispecific IgG

(115) Bispecific antibodies that were shown to bind the respective human and macaque orthologs in FACS were ranked on apparent affinity for both orthologs in FACS. Therefore, the stable cell lines expressing the respective orthologs (Table 1) were harvested and diluted to 10.sup.6 cells/ml in FACS buffer (PBS/0.5% BSA/0.5 mM EDTA). Cells were centrifuged for 2 minutes at 300 g at 4° C. Supernatant was discarded by inverting the plate(s). 50 μl of each IgG sample, in a 11-step, 2-fold dilution series ranging from 10 to 0.01 μg/ml, was added and incubated for 1H on ice. Cells were centrifuged once, supernatant was removed and cells were washed twice with 150 μl of FACS buffer. 50 μl diluted 1:400 goat anti human IgG PE (Invitrogen) was added and incubated for 30 minutes on ice in the dark. After adding FACS buffer, cells were centrifuged once, supernatant was removed and cells were washed twice with FACS buffer. Cells were analyzed on a FACSCanto Flow cytometer (Becton and Dickinson) in a HTS setting. Binding of the antibodies to cells was assessed by measuring the mean fluorescence intensity (MFI) of the stained cell population. Antibodies were considered to bind their target when the MFI was at least five-fold that of the same cell population stained with a (negative control) non-binding antibody (directed to tetanus toxoid).

(116) Binning huLAG-3 specific Fab arms present in LAG-3×LAG-3 bivalent IgG on domain specificity huLAG-3 binding clones in bivalent IgG format were tested for domain specificity in FACS on HEK293T cells that were transiently transfected with five different pIRES-Neo3 mouse/human LAG-3 hybrid expression constructs, a FL mouse LAG-3 pIRES-Neo3 expression construct (see amino acid insert sequences below) or the pIRES-Neo3_huLAG-3 expression construct used for generation of stable huLAG-3 expressing Freestyle 293F cells (Table 1). The same FACS protocol was used as described above during specificity analysis of the antibody panel. For generation of the hybrid constructs the extracellular domain of mouse and human LAG-3 was divided in 5 domains; 4 Ig-like domains based on Uniprot reference sequences P18627 (huLAG-3) and Q61790 (moLAG-3) and 1 hinge domain from end of Ig-like domain 4 to the transmembrane domain. The following amino acid insert sequences were cloned into pIRES-Neo3 (FIG. 4) via NheI/EcoRI; Text in bold is the signal peptide. Underscored text is the sequence identical to human LAG-3. Text in Italics represent the transmembrane and intracellular domain sequences.

(117) TABLE-US-00006 Amino acid sequence full length mouse LAG-3 insert. (SEQ ID NO: 6) MREDLLLGFLLLGLLWEAPVVSSGPGKELPVVWAQEGAPVHLPCSLK SPNLDPNFLRRGGVIWQHQPDSGQPTPIPALDLHQGMPSPRQPAPGR YTVLSVAPGGLRSGRQPLHPHVQLEERGIARGDFSLWLRPALRTDAG EYHATVRLPNRALSCSLRLRVGQASMIASPSGVLKLSDWVLLNCSFS RPDRPVSVHWFQGQNRVPVYNSPRHFLAETFLLLPQVSPLDSGTWGC VLTYRDGFNVSITYNLKVLGLEPVAPLTVYAAEGSRVELPCHLPPGV GTPSLLIAKWTPPGGGPELPVAGKSGNFTLHLEAVGLAQAGTYTCSI HLQGQQLNATVTLAVITVTPKSFGLPGSRGKLLCEVTPASGKERFVW RPLNNLSRSCPGPVLEIQEARLLAERWQCQLYEGQRLLGATVYAAES SSGAHSARRISGDLKGGHLVLVLILGALSLFLLVAGAFGFHWWRKQL LLRRFSALEHGIQPFPAQRKIEELERELETEMGQEPEPEPEPQLEPE PRQL Amino acid sequence mo/huLAG-3 chimeric insert A (Full length mouse LAG-3 sequence in which the mouse signal peptide and Ig-like domain 1 is replaced by the human signal peptide and Ig-like domain 1). (SEQ ID NO: 7) MWEAQFLGLLFLQPLWVAPVKPLQPGAEVPVVWAQEGAPAQLPCSPT IPLQDLSLLRRAGVTWQHQPDSGPPAAAPGHPLAPGPHPAAPSSWGP RPRRYTVLSVGPGGLRSGRLPLQPRVQLDERGRQRGDFSLWLRPARR ADAGEYRAAVHLRDRALSCRLRLRLGQASMIASPSGVLKLSDWVLLN CSFSRPDRPVSVHWFQGQNRVPVYNSPRHFLAETFLLLPQVSPLDSG TWGCVLTYRDGFNVSITYNLKVLGLEPVAPLTVYAAEGSRVELPCHL PPGVGTPSLLIAKWTPPGGGPELPVAGKSGNFTLHLEAVGLAQAGTY TCSIHLQGQQLNATVTLAVITVTPKSFGLPGSRGKLLCEVTPASGKE RFVWRPLNNLSRSCPGPVLEIQEARLLAERWQCQLYEGQRLLGATVY AAESSSGAHSARRISGDLKGGHLVLVLILGALSLFLLVAGAFGFHWW RKQLLLRRFSALEHGIQPFPAQRKIEELERELETEMGQEPEPEPEPQ LEPEPRQL Amino acid sequence mo/huLAG-3 chimeric insert B (Full length mouse LAG-3 sequence in which the mouse Ig-like domain 2 is replaced by the human Ig-like domain 2). (SEQ ID NO: 8) MREDLLLGFLLLGLLWEAPVVSSGPGKELPVVWAQEGAPVHLPCSLK SPNLDPNFLRRGGVIWQHQPDSGQPTPIPALDLHQGMPSPRQPAPGR YTVLSVAPGGLRSGRQPLHPHVQLEERGLQRGDFSLWLRPALRTDAG EYHATVRLPNRALSCSLRLRVGQASMTASPPGSLRASDWVILNCSFS RPDRPASVHWFRNRGQGRVPVRESPHHHLAESFLFLPQVSPMDSGPW GCILTYRDGFNVSITYNLKVLGLEPVAPLTVYAAEGSRVELPCHLPP GVGTPSLLIAKWTPPGGGPELPVAGKSGNFTLHLEAVGLAQAGTYTC SIHLQGQQLNATVTLAVITVTPKSFGLPGSRGKLLCEVTPASGKERF VWRPLNNLSRSCPGPVLEIQEARLLAERWQCQLYEGQRLLGATVYAA ESSSGAHSARRISGDLKGGHLVLVLILGALSLFLLVAGAFGFHWWRK QLLLRRFSALEHGIQPFPAQRKIEELERELETEMGQEPEPEPEPQLE PEPRQL Amino acid sequence mo/huLAG-3 chimeric insert C (Full length mouse LAG-3 sequence in which the mouse Ig-like domain 3 is replaced by the human Ig-like domain 3). (SEQ ID NO: 9) MREDLLLGFLLLGLLWEAPVVSSGPGKELPVVWAQEGAPVHLPCSLK SPNLDPNFLRRGGVIWQHQPDSGQPTPIPALDLHQGMPSPRQPAPGR YTVLSVAPGGLRSGRQPLHPHVQLEERGLQRGDFSLWLRPALRTDAG EYHATVRLPNRALSCSLRLRVGQASMIASPSGVLKLSDWVLLNCSFS RPDRPVSVHWFQGQNRVPVYNSPRHFLAETFLLLPQVSPLDSGTWGC VLTYRDGFNVSIMYNLTVLGLEPPTPLTVYAGAGSRVGLPCRLPAGV GTRSFLTAKWTPPGGGPDLLVTGDNGDFTLRLEDVSQAQAGTYTCHI HLQEQQLNATVTLAVITVTPKSFGLPGSRGKLLCEVTPASGKERFVW RPLNNLSRSCPGPVLEIQEARLLAERWQCQLYEGQRLLGATVYAAES SSGAHSARRISGDLKGGHLVLVLILGALSLFLLVAGAFGFHWWRKQL LLRRFSALEHGIQPFPAQRKIEELERELETEMGQEPEPEPEPQLEPE PRQL Amino acid sequence mo/huLAG-3 chimeric insert D (Full length mouse LAG-3 sequence in which the mouse Ig-like domain 4 is replaced by the human Ig-like domain 4). (SEQ ID NO: 10) MREDLLLGFLLLGLLWEAPVVSSGPGKELPVVWAQEGAPVHLPCSLK SPNLDPNFLRRGGVIWQHQPDSGQPTPIPALDLHQGMPSPRQPAPGR YTVLSVAPGGLRSGRQPLHPHVQLEERGIARGDFSLWLRPALRTDAG EYHATVRLPNRALSCSLRLRVGQASMIASPSGVLKLSDWVLLNCSFS RPDRPVSVHWFQGQNRVPVYNSPRHFLAETFLLLPQVSPLDSGTWGC VLTYRDGFNVSITYNLKVLGLEPVAPLTVYAAEGSRVELPCHLPPGV GTPSLLIAKWTPPGGGPELPVAGKSGNFTLHLEAVGLAQAGTYTCSI HLQGQQLNATVTLAIITVTPKSFGSPGSLGKLLCEVTPVSGQERFVW SSLDTPSQRSFSGPWLEAQEAQLLSQPWQCQLYQGERLLGATVYAAE SSSGAHSARRISGDLKGGHLVLVLILGALSLFLLVAGAFGFHWWRKQ LLLRRFSALEHGIQPFPAQRKIEELERELETEMGQEPEPEPEPQLEP EPRQL Amino acid sequence mo/huLAG-3 chimeric insert E (Full length mouse LAG-3 sequence in which the mouse Ig-like domain 4 including the hinge up to the transmembrane sequence is replaced by the human Ig-like domain 4 including the hinge). (SEQ ID NO: 11) MREDLLLGFLLLGLLWEAPVVSSGPGKELPVVWAQEGAPVHLPCSLK SPNLDPNFLRRGGVIWQHQPDSGQPTPIPALDLHQGMPSPRQPAPGR YTVLSVAPGGLRSGRQPLHPHVQLEERGIARGDFSLWLRPALRTDAG EYHATVRLPNRALSCSLRLRVGQASMIASPSGVLKLSDWVLLNCSFS RPDRPVSVHWFQGQNRVPVYNSPRHFLAETFLLLPQVSPLDSGTWGC VLTYRDGFNVSITYNLKVLGLEPVAPLTVYAAEGSRVELPCHLPPGV GTPSLLIAKWTPPGGGPELPVAGKSGNFTLHLEAVGLAQAGTYTCSI HLQGQQLNATVTLAIITVTPKSFGSPGSLGKLLCEVTPVSGQERFVW SSLDTPSQRSFSGPWLEAQEAQLLSQPWQCQLYQGERLLGAAVYFTE LSSPGAQRSGRAPGALPAGHLVLVLILGALSLFLLVAGAFGFHWWRK QLLLRRFSALEHGIQPFPAQRKIEELERELETEMGQEPEPEPEPQLE PEPRQL
Tables 3 and 4 show the bins to which each LAG-3×LAG-3 bivalent IgG was assigned based on domain specificity.
Reference Antibodies

(118) Antibodies that inhibit the function of PD-1 and LAG-3 are known in the art. The information with regard to the anti-PD-1 antibody Nivolumab was generated based on the information disclosed in CA2607147 and was expressed in CHO-S cells. The anti-LAG-3 antibody 25F7 was regenerated based on information provided in WO2010/019570A2 (Medarex. Inc) recloned in an IgG1 backbone and expressed in 293F Freestyle cells.

(119) LAG-3 Blockade Reporter Assay

(120) The LAG-3 blockade reporter assays were performed using the LAG-3 blockade reporter assay developed by Promega that uses a two cell system based on Raji cells that expressed MHCII and a Jurkat/NFAT-RE Reporter Cell Line overexpressing LAG-3. Activation of Jurkat cells is controlled via superantigen SED, Staphylococcal Enterotoxins D. LAG-3 Jurkat Effector cells were provided by Promega in a Cell Propagation Model (CPM) format and propagated in RPMI 1640 (+L-glutamine, 10% heat inactivated FBS, 100 μM MEM non-essential amino acids, 1 mM sodium pyruvate, 200 μg/ml Hygromycin and 500 μg/ml G418). Raji cells were propagated in RPMI 1640 (+L-glutamine and 10% heat inactivated FBS). Cells growing in logarithmic phase were harvested and resuspended in RPMI 1640 containing 1% heat inactivated FBS) at a concentration of 2×10.sup.6 cells/ml Raji and 4×10.sup.6 cells/ml for Jurkat/NFAT-RE cells. Next, 25 μl Jurkat/NFAT-RE cell suspension was added to the inner wells of a 96 well plate (Corning, Cat #3917). Next, 25 μl test antibody in assay medium (RPMI 1640 containing 1% FBS) in a serial dilution (starting concentration 25 μg/ml) was added to each well. Each plate contained a serial dilution of negative (PG1337) and positive control antibody 25F7 that served as reference controls. Finally 25 μl of a 1:1 mixture of Raji cells and (100 ng/ml) SED (Toxin Technologies) were added. Plates were incubated for 6 H at 37° C., 5% CO, in 95% relative humidity. 40 μl of luciferase (Bio-Glo Luciferase Assay System, cat. no. G794L) was added the next day and the amount of luciferase activity was measured using aBioTek Synergy 2 Multi-Mode Microplate Reader. LAG-3 antibodies were screened in bivalent format to determine their LAG-3 blocking capacity (FIG. 11).

(121) PD-1/PD-L1 Blockade Reporter Assay

(122) The PD-1/PD-L1 blockade reporter assays used were developed by Promega and are based on a two cell system; CHO cells expressing PD-L1, and a T cell activator and a Jurkat/NFAT-RE Reporter Cell Line overexpressing PD-1. The PD-1/PD-L1 blockade reporter assays were performed using the thaw and use format of Promega. PD-L1 expressing cells (cat. no. C187103) were thawed in 14.5 ml Cell Recovery Medium (DMEM/F12 containing 10% FBS). Next, 50 μl cell suspension was added to the inner wells of a 96 well half area plate (Corning, cat. no. 3688). Plates were incubated overnight at 37° C., 5% CO, in 95% relative humidity. Next day, culture medium was removed and 20 μl test antibody in assay medium (RPMI 1640 containing 4% FBS) in a serial dilution (starting concentration 10 μg/ml) was added to each well. Each plate contained a serial dilution of negative (Ctrl Ab) and positive control antibody (Nivolumab) that served as reference controls. PD-1 effector cells (cat no. C187105) were thawed in 5.9 ml Assay medium and 20 μl cell suspension was added to each well. Plates were incubated for 6 H or overnight at 37° C., 5% CO, in 95% relative humidity. 40 μl of luciferase (Bio-Glo Luciferase Assay System, cat. no. G794L) was added the next day and the amount of luciferase activity was measured using aBioTek Synergy 2 Multi-Mode Microplate Reader. Potency was measured as luciferase activity in comparison to the negative control antibody.

(123) PBMC Isolation

(124) Human whole blood was obtained from buffy coats (Sanquin) and was diluted 1:1 with PBS. Leucosep tubes (Greiner Bio-One cat. no. 227 290) were filled with 17.5 m Ficoll-Paque Plus (Amersham Biosciences cat. no. 17-1440-02) warmed at room temperature (RT). Ficoll-Paque Plus was spun down for 30 seconds at 1000×g at RT. 30 ml of diluted whole blood was poured on top. The tubes were spun at 1000×g for 10 minutes at RT and the mononuclear PBMC interface was harvested, washed twice in PBS and resuspended in 250 μl PBS. The PBMCs were counted and readjusted to 1×106/ml in tissue culture medium (DMEM with 10% FCS) and frozen down by adding an equal volume of ice-cold freeze medium (80% culture medium/20% DMSO). Cells were stored in 1 ml aliquots at −150° C. until further use.

(125) SEB Assay

(126) The functional activity of the bispecific antibodies was determined by using PBMCs stimulated by Staphylococcus enterotoxin B (SEB). SEB specifically activates T cells expressing the Vβ3 and Vβ8 T cell receptor chain. PBMCs from 3 donors is thawed, washed, counted and resuspended in culture medium (RPMI1640 plus 10% heat inactivated FBS) to a concentration of 2 10.sup.6 cells/ml. Cells were seeded in flat bottom 96-well plates (2×10.sup.5 cells/well) in the presence of SEB (2000 or 125 ng/ml). Antibody serial dilutions starting at 20 g/ml were added. Each plate contained a serial dilution of negative (Ctrl Ab) and positive control antibody (nivolumab and LAG-3(25F7) that served as reference controls. Cells were stimulated for 3 days at 37° C., 5% CO2 in 95% relative humidity prior to being tested for cytokine secretion and/or cell surface expression of antigens.

(127) Cytokine Assays

(128) ELISA: After stimulation of T-cells or PBMCs at various times, plates were centrifuged and media was removed. Cytokine levels were detected by AlphaLISA in accordance with the manufacturer's instructions (Perkin Elmer). Concentrations were calculated based on the standard curve.

(129) Luminex assay: Another method used to determine cytokine production in vitro was using luminex analysis developed by eBioscience. Levels of IL-2 were measured in culture supernatants following ranufacturers' instructions. Results were analyzed by eBioscience analysis software.

(130) Screening of the PD-1 Antibody Panel

(131) VH from the PD-antibody panel were produced in 24 well format and tested as bivalent antibodies in a semi log serial titration (starting concentration 10 μg/ml) in the PD-1/PD-L1 blockade reporter assay to rank the antibodies for blocking potency in comparison to Nivolumab. Based on the activity data antibodies were selected from the PD-1 antibody panel for the subsequent PD1×LAG-3 bispecific screen. The activity of the selected candidates in the reporter assay is shown in Table 2. The PD-1 Fab panel was composed of functional activity variants within two antibody clusters i.e. A and B.

(132) Screening PD1×LAG-3 Antibody Panel

(133) VH from the PD-1 and LAG-3 antibody panel were recloned into the charged engineered Fc-silenced vectors such that upon expression of the antibody heavy chains hetero dimerisation of heavy chains is forced resulting in the generation of bispecific antibodies after transfection. The PD-1 Fab arms were cloned in the MV1625 vector whereas the LAG-3 Fab arms were recloned in the MV1624 vector. Fifteen LAG-3 Lab arms representing the different bins (Table 3) were combined with three PD-1 Fab arms (MF6226, MF6930 and MF6256) displaying a range of PD-1 blocking activities (Table 2). Bispecific LAG-3×PD-1 antibodies (Table 4), their bivalent parental LAG-3 antibodies and negative control antibodies were tested for their capacity to activate T cells by a serial dilution of SFB (2000-500-125-31-8.Math.2 ng/mL). FIG. 12 shows the activity of two LAG-3 bivalent antibodies in comparison to LAG-3×PD-1 antibodies with different PD-1 affinity on PBMC cells stimulated with 2 μg/ml SEB. The activity is represented as stimulation index. Each IL-2 value is compared to the negative control antibody to determine the SI (SI, IL-2 of 2 means 200% increase in IL-2 production when compared to the control antibody). LAG-3×PD-1 bispecific antibodies induced more IL-2 in the SEB assay in comparison to the bivalent LAG-3 antibodies. Selections of bispecific antibodies, representing each bin, were subsequently screened in a serial dilution in a SEE assay whereby PBMC were stimulated with 2 μg/ml SEB (FIG. 13). The majority of bispecific LAG-3×PD-1 antibodies were more potent than the parental PD-1 bivalent antibody or reference LAG-3 antibody in inducing IL-2 release. LAG-3×PD-1 bispecific antibodies that bound other domains than the MHC Class II interacting domain i.e. domain 3 and 4 were also more potent than the parental PD-1 bivalent antibody or reference LAG-3 antibody in inducing IL-2 release.

Example 4. Screening of a PD1×LAG-3 Antibody Panel

(134) The Fab of antibody 25117, a LAG-3 antibody, was cloned and produced in a human IgG1 format as PG7431 as described above in Example 3. The variable domain of 25F7 was also cloned into a monovalent LAG-3 antibody format as PB22283 wherein the first arm comprised the 25F7 variable domain and the second arm comprised a tetanus toxoid binding variable domain (MF1337). Binding of these antibodies are shown in FIG. 14. PG1337P300 is a control antibody that is not expected to bind to the cells and binds tetanus toxoid.

(135) Binding of these antibodies was compared with the bivalent antibody PG7116 which has two variable domains with MF7116 and a monovalent LAG-3 antibody format wherein the first arm comprised the variable domain of MF7116 and the second arm comprised the tetanus toxoid variable domain comprising MF1337.

(136) Binding of the antibodies in bivalent form is similar as can be seen by comparing the binding of PG7431 with PG7116 in the left hand panel where the antibodies were titrated on Freestyle 293F_huLAG-3 (293FF LAG-3). The binding of the monovalent variable domain of 25F7 is similar to the binding in bivalent form (compare PB22283 with PG7431 in FIG. 15, right hand panel, where the antibodies were titrated on activated T-cells). Binding of monovalent variable domain with MF7116 is reduced when compared to the bivalent antibody PG7116 (compare P121775 with PG7116, FIG. 15, left hand panel).

(137) Accordingly, LAG-3 IgGs were tested in binding assays in bivalent and monovalent formats, and were compared to a 25F7 (PG7431) antibody. LAG-3 antibodies in bivalent format showed similar binding curves as the benchmark antibodies. Monovalent LAG-3×TT antibodies typically showed reduced binding activity compared to the bivalent molecules.

(138) In order to further characterize PD-1×LAG-3 bispecific antibodies, a panel of bispecific PD-1×LAG-3 antibodies was created as set out in Table 5 and functionally tested in a PD-1×LAG-3 reporter assay.

(139) In the PD1/Lag3 reporter assay, Jurkat Effector Cells as described above (modified to overexpress PD-1 and LAG-3) and target Raji Cells (modified to overexpress PD-L1), SED and LAG-3 antibodies are mixed and incubated. The Jurkat cell line contains a luciferase reporter gene that can become activated through the NFAT (nuclear factor of activated T-cells) pathway. Interaction of the MHCII with LAG-3 will inhibit this signal and blocking the MHCII/LAG-3 interaction by biologics can release the signal as well as the blocking of the PD-1/PD-L1 signal by biologics on the PD-1 receptor.

(140) The PD-1×LAG-3 bispecific antibodies were titrated in the PD-1/LAG-3 reporter assay and the activity was compared with a bivalent LAG-3 antibody 25F7 and the bivalent PD-1 antibody (both as described above in Example 3). The bispecific antibodies facilitate the activation of the Jurkat cells at significantly lower concentrations than the monospecific bivalent control antibodies. The activity is comparable to the activity when the two control antibodies are combined, in spite of the fact that the bispecific antibodies are monovalent for each of the targets which, as demonstrated above, can reduce the binding of a monovalent LAG-3 antibody to LAG-3 (see FIG. 16).

(141) A representative example of the reporter assay screening is presented in FIG. 17: various combinations of LAG-3 and PD-1 variable domains are depicted.

(142) Panel A shows bispecific antibodies with variable domains that bind PD-1 and LAG-3 and that block the binding of natural ligands to the receptor (respectively PD-L1/L2 and MHC class II). The particular bispecific antibodies depicted in panel A allow the activation of the Jurkat reporter cell than a reference having the two monospecific bivalent antibodies to PD-1 and 25F7. Panel B shows bispecific antibodies having the same LAG-3 binding variable domain but now in combination with a PD-1 variable domain that binds PD-1 but that does not block PD-1/PD-L1 signaling. It is clear that the activity of the bispecific antibody in panel A is mediated by at least the arm that binds PD-1 and that blocks PD-1/PD-L1 signaling. Panel C shows the reverse example, the PD-1 arm of panel A in combination with LAG-3 binding variable domains that bind, but that do not block MHC II/LAG-3 signaling. The results shows that the activity of the bispecific antibody depicted in Panel A is mediated at least by a LAG-3 binding arm that blocks the binding of MHC class II to LAG-3.

(143) FIG. 18 summarizes the results of the reporter screening and indicates the area under the curve relative to control.

(144) SEB assays were carried out as described in Example 3. FIG. 19 sets out a representative example of an SEB titration assay of the PD-1/LAG-bispecific antibodies as described herein. The figure shows the activity of two LAG-3 bivalent antibodies in comparison to PD-1×LAG-3 antibodies with different PD-1 affinity on PBMC cells stimulated with 2 μg/ml SE.

(145) Panel A shows the result of bispecific antibodies with variable domains that bind PD-1 and LAG-3 and that block the binding of natural ligands to the respective receptors. These antibodies facilitate the production of IL-2 in the PBMC cells. Panel B shows the results with bispecific PD-1×LAG-3 antibodies that have a PD-1 variable domain that binds PD-1 but that does not block the binding of PD-L1 to the receptor. The LAG-3 variable domain blocks the binding of LAG-3 to MHC II. Panel C shows the results with bispecific antibodies with a variable domain that binds LAG-3 but that does not block the binding of LAG-3 to MHC II. The variable domain that binds PD-1 blocks the binding of PD-1 to PD-L1.

(146) A summary of the screening of the bispecific panel set out in Table 5 is depicted in FIGS. 20A-20C. The PD-1 variable domains were ranked top to bottom depending on the activity of the bivalent antibody in the reporter assay (upper panel) and SEB assay based on 2 donors (middle and lower panel) with the PD-1 variable domain with the highest activity in the reporter assay at the top. The LAG-3 variable domains are ranked high to low in the reporter assay (upper panel) and SEB assay based on 2 donors (middle and lower panel) with the variable domain with the highest activity in the bivalent antibody format in the first column.

(147) The PD-1 arms within bispecific PD-1×LAG3 antibodies were ranked by determining how many times a given arm within a PD-1×LAG3 bispecific appeared in the top 15% of bispecific antibodies based on percent AUC as compared to positive control in: 1) Reporter assay; 2) SEB screening donor 1 (IL-2 data); 3) SEB screening donor 2 (IL-2 data). See FIGS. 21 and 23. This ranking is illustrated in the two right most columns of FIG. 23.

(148) It can be seen that clones with PD-1 arms having a variable domain with a VH of MF6974 or a VH of MF6076 were ranked highest based on the above criteria with most of the LAG-3 arms in the Reporter and SEB assays.

(149) The same approach was taken for bispecific antibodies carrying a specific LAG-3 arms, which were also scored on the basis of how many times they were present in the top 15% (those arms with same score in the top 15% were further ranked by using the top 25% scores). The final ranking is set out in FIG. 22.

(150) Several bivalent, monospecific antibodies (in IgG format) that bind LAG3 domain 1, according to mouse/human LAG-3 hybrid binning experiments described above, scored highest based on AUC percent of a positive control in a reporter assay. However, the bispecific antibodies binding PD1 and LAG3 that were ranked the highest based on reporter assays and in the SEB assay in IL-2 induction were those that domain 2 (as determined according to the mouse/human LAG-3 hybrid experiments).

(151) This is summarized in FIG. 24. Of the top eight bispecifics as ranked based on the PD1/LAG3-reporter assay and SEB, six comprised a LAG3 arm that binds domain 2 (as determined according to the mouse/human LAG-3 hybrid experiments). The activity of the LAG3 arms (on the basis of AUC percent of control) from these 6 bispecifics as determined by LAG3 reporter assay in bivalent monospecific format was typically lower than that of LAG3 arms tested in the same format which were determined to bind domain 1.

Example 5

(152) Bispecific PD-1×LAG-3 Antibodies Enhance IFNγ Production by CD14+ T Cells in a Mixed Lymphocyte Reaction.

(153) Mixed lymphocyte reaction (MLR) assays are commonly used to understand the effects of antibodies on T-cell activation and proliferation. Such assays aid understanding of whether such compounds will affect the potential of T cells to mount a response in the tumor microenvironment. Here we used an allogeneic MLR protocol with immature DCs to determine the ability of bispecific PD-1×LAG-3 antibodies to enhance IFNγ production by CD14+ T cells, compared with that of benchmark reference antibodies. The responsiveness of the T cells was quantified by measuring the levels of IFNγ in culture supernatant.

(154) To this end, human peripheral blood mononuclear cells (PBMCs) from healthy donors were prepared from buffy coats. Immature monocyte-derived dendritic cells (Mo-DCs) were prepared by isolating CD14+ cells (EasySep Stemcell, lot no. 16C69672) using magnetic activated cell sorting (MACS) and culturing these in differentiation medium for seven days. Responder T cells derived from a different donor to that used for the Mo-DCs were prepared from cryopreserved PBMCs on the day required, using a T-cell isolation kit (EasySep Stemcell, lot no. 16D70573) to obtain untouched T cells. Six separate MLRs were performed to provide biological replication.

(155) For the assay, 1×10.sup.4 immature Mo-DCs were co-cultured with 1×10.sup.5 CD14+ T cells for 4 days, in the presence or absence of test antibody at an end concentration of 10 μg/mL. Cultures were performed in triplicate. Supernatants were collected at the end of the culture period and assessed for IFNγ by ELISA (R&D BioTechne, lot no. 342687) according to the manufacturer's instructions with plates read at 450 nm.

(156) Results

(157) The MLR study comprised experimental groups of a LAG3/PD1 bispecific (PB15307=MF7137 (LAG3) and MF6930 (PD1)) and LAG3 isotype control group (mono-specific antibody against a LAG-3 (bivalent monospecific antibody PG7431), Tetanus and mIgG1). Single cell controls and a vehicle control group were also included.

(158) Cultures were performed in triplicate to provide technical replicates. At the end of the 4-day culture period supernatant was collected and ELISAs performed to assess effects of the antibodies on the production of IFN-y, according to manufacturer's instructions with plates read at 450 nM.

(159) CD14+ cells were sorted on DO and cultured for 7 days, immature DCs were used on D7 and mature DCs were obtained by culturing for an additional 3 days in maturation medium. CD14 positivity was assessed at DO to confirm purity of the initial sort, and in immature and matured DCs at D7 and D10, respectively, to confirm downregulation of CD14 to indicate differentiation to Mo-DCs (data not shown). Viability and activation markers (CD80, CD83 and CD86) were also assessed on both immature and matured DCs to confirm differentiation and maturation. Mo-DCs (immature or mature) were cultured with responder T cells for 4 days before supernatant was collected and ELISAs performed to assess effects of the test antibodies on production of IFN-y. In the mature MLR (mMLR) the donor variance was such that the data were normalized to vehicle control for each donor (raw data and normalized.

(160) In FIG. 25 the results of the test are shown. The bispecific antibody specific for PD-1/LG-3 out-performed IFN-y production by CD14+ T cells in MLR significantly over the control and LAG3 surrogate monospecific antibody.

(161) TABLE-US-00007 TABLE 1 Expression constructs for each target that were used for DNA immunization (pVAX1 vector based) and for generation of stable Freestyle 293F or CHO-S cell lines (pIRES-neo3 vector based or similar) Target Vectors Stable cell line PD-1 pVAX l_huPD-1 NA pIRES-neo3_huPD-1 CHO-S_huPD-1 pIRES-neo3_maPD-1 CHO -S_maPD-1 LAG-3 pVAXl_huLAG-3 NA pVAX1_raLAG-3 NA pIRES-neo3_huLAG-3 Freestyle 293F_huLAG-3 pIRES-neo3_maLAG-3 Freestyle 293F_maLAG-3 hu = human, ma = macaque, ra = rat, NA = not applicable

(162) TABLE-US-00008 TABLE 2 Functional activity of PD-1 Fab arms as measured in the PD-1/PD-L1 blockade reporter assay as a bivalent antibody in comparison to the positive control Nivolumab. Variants of the same cluster (B) that displayed different of PD-1 blocking activity were tested. SEQ ID % Activity of NOS. Clone CDR3 Cluster pos control SEQ ID  MF6226 GGYSGYGGDSFDL A 47.77614647 NO: 111 SEQ ID MF6256 GTVEATLLFDF B 57.85260834 NO: 112 SEQ ID MF6930 GTVEATLLFDY B 51.50445453 NO: 113

(163) TABLE-US-00009 TABLE 3 Panel of LAG-3 Fab arms describing binning based on FACS profiles, domain binding and LAG-3 blocking activity as bivalent antibody. SEQ ID LAG-3 NO: ID CDR3H VH Bin block SEQ ID 7111 IPLTGEFDY VH4-59 D1 Yes NO: 124 SEQ ID 7165 GGTYYYGSGSYYTLDY VH1-24 D1 Yes NO: 142 SEQ ID 7116 DGDNWDVFDI VH3-30 D1 Yes NO: 143 SEQ ID 7100 ERGWDVFDI VH3-30 D1 Yes NO: 144 SEQ ID 7137 GGTYYYGSGSYYTLDF VH1-24 D1 Yes NO: 145 SEQ ID 7518 DGSGWDDFDY VH1-18 D1 + D4 Yes NO: 146 SEQ ID 7134 EPNWGVYFDY VH7-4-1 D2 Yes NO: 147 SEQ ID 7146 DREVGAIYYFDY VH1-69 D2 Yes NO: 148 SEQ ID 7142 ERDIGSLYYFDS VH1-69 D2 Yes NO: 149 SEQ ID 7185 DREMFTLYFFDQ VH1-69 D2 Yes NO: 150 SEQ ID 7136 DSTYYYTSGSYSVFDY VH3-23 D2 No NO: 151 SEQ ID 7118 VPAAATPSGTYYWIFDL VH3-23 D3 No NO: 117 SEQ ID 7443 DTSTWQRGGYKAFDY VH3-23 D3 No NO: 152 SEQ ID 7167 DRGYDYSGSYHNWFDP VH3-23 D4 No NO: 153 SEQ ID 7515 RPGPALGDLDS VH1-18 D4 No NO: 154 SEQ ID 7444 DTGQSWSNYYHAFDY VH3-23 hu/mo cross- No NO: 155 reactive-  hu D3

(164) TABLE-US-00010 TABLE 4 Panel of LAG-3 Fab arms describing binning based on FACS profiles and domain binding. ID CDR3H VH Bin 7096 DLLYKWNYVEGFDI VH4-59 D1 7097 DLLYKWNYVEGFDI VH4-59 D1 7106 DKAVAGLYYFDS VH1-69 D2 7118 VPAAATPSGTYYWIFDL VH3-23 D3 7120 ERELGALYAFDI VH1-69 D2 7133 DRETGTLYYFDY VH1-69 D2 7139 DRAIGTLYYFDY VH1-69 D2 7144 DRDSGGLYYFDS VH1-69 D2 7524 GSILAAQMWGDI VH1-18 hu/mo cross- reactive-mo 1 SEQ ID No: ID CDR3H VH Bin SEQ ID 7096 DLLYKWNYVEGFDI VH4-59 D1 NO: 114 SEQ ID 7097 DLLYKWNYVEGFDI VH4-59 D1 NO: 115 SEQ ID 7106 DKAVAGLYYFDS VH1-69 D2 NO: 116 SEQ ID 7118 VPAAATPSGTYYWIFDL VH3-23 D3 NO: 117 SEQ ID 7120 ERELGALYAFDI VH1-69 D2 NO: 118 SEQ ID 7133 DRETGTLYYFDY VH1-69 D2 NO: 119 SEQ ID 7139 DRAIGTLYYFDY VH1-69 D2 NO: 120 SEQ ID 7144 DRDSGGLYYFDS VH1-69 D2 NO: 121 SEQ ID 7524 GSILAAQMWGDI VH1-18 hu/mo cross- NO: 122 reactive-mo 1

(165) TABLE-US-00011 TABLE 5 Overview PB numbers and their MF composition PD-1 LAG-3 MF6930 MF6226 MF6256 MF7111 PB15292 PB16336 PB15254 MF7116 PB15296 PB16367 PB15258 MF7100 PB15289 PB16369 PB15251 MF7137 PB15307 PB16365 PB15269 MF7518 PB15383 PB16364 PB15347 MF7134 PB15305 PB16337 PB15267 MF7146 PB15313 PB16338 PB15275 MF7142 PB15311 PB16339 PB15273 MF7165 PB15317 PB16366 PB15279 MF7185 PB15363 PB16340 PB15359 MF7136 PB15306 PB16341 PB15268 MF7118 PB15297 PB16342 PB15259 MF7443 PB15369 PB16343 PB15333 MF7444 PB15403 PB16346 PB15393 MF7167 PB15318 PB16344 PB15280 MF7515 PB15380 PB16345 PB15344

(166) TABLE-US-00012 TABLE 5 Overview of LAG-3 arms and PD-1 arms and which heavy chains the variable domains are associated with in a bispecific antibody Target MF Target MF LAG -3 7096 PD -1 5743 7097 6076 7100 6225 7106 6227 7111 6930 7116 6932 7118 6935 7120 6974 7133 6983 7134 7137 7139 7142 7144 7167 7185 7444 7518 7524