The CC′ loop of IgV domains of the immune checkpoint receptors, plays a key role in receptor:ligand affinity modulation

Antibodies targeting negative regulators of immune checkpoints have shown unprecedented and durable response against variety of malignancies. While the concept of blocking the negative regulators of the immune checkpoints using mAbs appears to be an outstanding approach, their limited effect and several drawbacks, calls for the rational design of next generation of therapeutics. Soluble isoforms of the negative regulators of immune checkpoint pathways are expressed naturally and regulate immune responses. This suggests, affinity-modified versions of these self-molecules could be effective lead molecules for immunotherapy. To obtain better insights on the hotspot regions for modification, we have analysed structures of 18 immune receptor:ligand complexes containing the IgV domain. Interestingly, this analysis reveals that the CC′ loop of IgV domain, a loop which is distinct from CDRs of antibodies, plays a pivotal role in affinity modulation, which was previously not highlighted. It is noteworthy that a ~5-residue long CC′ loop in a ~120 residue protein makes significant number of hydrophobic and polar interactions with its cognate ligand. The post-interaction movement of CC′ loop to accommodate the incoming ligands, seems to provide additional affinity to the interactions. In silico replacement of the CC′ loop of TIGIT with that of Nectin-2 and PVR followed by protein docking trials suggests a key role of the CC′ loop in affinity modulation in the TIGIT/Nectin pathway. The CC′ loop appears to be a hotspot for the affinity modification without affecting the specificity to their cognate receptors.

The concept of blocking the negative regulators of the immune system (checkpoint blockade) to control/eradicate cancer and its durable response over the plethora of cancer types, has revolutionised the way cancer is treated today. Here, the reactivation of the immune system to fight cancer lies at the core of its philosophy. The involvement of co-stimulatory or inhibitory immune checkpoint receptors (ICRs) 1-3 , along with primary MHC-2 (Major Histocompatibility Complex-2) and TCR (T-cell receptor) signalling, is paramount for the activation or deactivation of T-cells. These immune checkpoint pathways like CTLA-4/CD28:B7-1/B7-2, CD226/TIGIT:Nectin-2/PVR and PD-1:PD-L1/PD-L2, hold considerable sway over the functional fate of the T-cells, B-cells, NK cells and so on 4 . Interestingly, tumour cells have been known to exploit some of these pathways by over-expressing the ligands of the inhibitory ICRs to escape immunosurviellance 5 . The blockade of signalling of these negative pathways through antibodies resulted in the reinvigoration of exhausted T-cells 6 , which eventually led to the regression of tumour size [7][8][9] .
Extraordinary amount of research on this conceptually novel approach and subsequent preclinical and clinical studies have resulted in the approval of effective monoclonal antibodies (mAbs) against inhibitory ICRs, as cancers therapeutics [10][11][12][13][14] , which was pioneered by the first-in-class mAb called Yervoy 15,16 . Immune checkpoint blockade via monoclonal antibodies became so influential in treating cancer that the Nobel prize for Physiology/ Medicine 2018 was awarded to James P. Allison and Tasuku Honjo 'for their discovery of cancer therapy by inhibition of negative immune regulation' . As the popularity of mAbs in cancer therapy grew, so did the reports of their drawbacks namely, high cost 17 , poor solid tumor infiltration of these mAbs 18 , moderate to severe adverse effects 19 and the observation that the mAb based therapy is responsive only in minority of patients 20 . Further, that several aspects of their association are quite different from each other. In antibodies, the CC′ loop from two associating IgV domains are always close to each other and are involved in the formation of heterodimeric hydrophobic core, which restricts relative orientation of two IgV domains. Interestingly, no such unique orientation is observed in the interactions of IgV domains of different ICR complexes. In fact, almost every ICR complex has a different binding orientation where the positions of CC′ loops and FG loops are variable ( Fig. 1c-g). This is illustrated well in the case of m2B4-mCD48 complex (PDB ID: 2PTT) where the CC′ loop of mCD48 is found near the vicinity of the FG loop of m2B4 and vice versa (Fig. 2d,g). In some cases, for example homodimeric NTB-A, two CC′ loops are close to each other, whereas the FG loops are far away 44 . These different binding orientations observed in ICRs point towards the varying degree to which the CC′ loop is involved in the interaction interface. In ICR complexes, the role of the front face, FG loop and the CC′ loop are much like the role of palm (front face), index-finger (FG loop) and thumb (CC′ loop) of a clapping hand. Hence, the FG loop and the CC′ loop flanking the front face act as clamps in order to hold the incoming binding partner. This analysis, together with existing mutagenesis studies (Supplementary Table 1), suggests that the CC′ loop makes vital interactions that fine-tune the affinity between the interacting IgV domains, whereas the FG loop and the front face provide the required specificity.
Based on these observations, we analysed 18 ICR complexes and it was observed that the CC′ loop of about 49% of the proteins analysed, contributed more than 10% to the total BSA of interaction interface of one IgV domain. This is an important statistic as the average length of the CC′ loop is only 5 residues long. Hence, it is interesting to observe how a ~5-residue long loop in a ~120 residue protein contributes more than 10% of the total BSA of the interaction interface in nearly half of the ICR complexes analysed. Additionally, along with the key BSA contributions, about 27% of the 33 proteins analysed, had hydrophilic interactions like hydrogen bonds and salt bridges contributed by the CC′ loop towards the interaction interface. It was also observed that about 24% of the proteins considered for this study, had more BSA contribution from CC′ loop than the FG loop towards the interaction interface (Table 1). These statistics hints at the considerable influence of CC′ loop in the interaction-interface of these complexes.
Post-interaction conformational changes or the induced fit of CC′ loop was another interesting feature that was observed. About 27% of the analysed ICRs showed deviation of more than 3 Å of the C α atom of at least one of the residues in the CC′ loop before and after interaction. This value is only 9% in the case of FG loop. Further, the extent of post-interaction movement of CC′ loop is significantly larger when compared to that of the FG loop where, maximum deviation of 9.5 Å and 5.6 Å have been observed for CC′ loop and FG loop respectively. This difference seems to indicate that the CC′ loop is subjected to more post-interaction movements to accommodate the incoming ligand than FG loop. It must be pointed out that alongside front face, the FG loop appears to play a pivotal role in providing the specificity of the ICR interactions. This is observed in TIGIT:TIGIT/Nectin-2/PVR pathway where a conserved 112 TYP 114 motif present on the FG loop of TIGIT helps in the lock-and-key interaction with its ligands Nectin-2 38,45 , Polio virus receptor (PVR) and itself 46 . Similarly, the FG loop of hCTLA-4 and hCD28 with a rigid 99 MYPPPY 104 motif provides specificity to their cognate receptors, B7-1 and B7-2 47 . Hence, www.nature.com/scientificreports www.nature.com/scientificreports/ it could be hypothesized while FG loop is involved in conferring specificity, CC′ loop's ability to undergo ligand induced conformational changes might be contributing to additional affinity.
As depicted in the Table 1, it is evident that the CC′ loop appears to contribute to ligand:receptor interactions in ICR complexes and in some cases, ligand induced conformational changes appear to be an important factor. In the case of PVR, it has also been shown that its ligands TIGIT and CD96 48 induce distinct CC′ loop movement upon complexation. The same CC′ loop is disordered in one of the two PVR:DNAM-1 complexes observed in the asymmetric unit 49 , suggesting the versatility of CC′ loop and its possible contribution to varied receptor:ligand binding affinities. Hence, based on the role of CC′ loop and intricacies of the post-interaction conformation changes in the CC′ loop, some interesting features were highlighted, 1) Significant post-interaction movement www.nature.com/scientificreports www.nature.com/scientificreports/ of CC′ loop 2) Loop to β-strand transition of CC′ loop upon interaction 3) Unusual conformation of CC′ loop leading to non-canonical IgV domain. In the following sections, we provide few examples of post-interaction conformational changes and two examples of unique variation in CC′ loop leading to non-canonical conformation that carve a binding pocket for small molecules.
Significant post-interaction movement in CC′ loop. In murine 2B4:CD48 complex (Fig. 2a), 2B4 is a member of signalling lymphocyte activation molecule (SLAM), and a receptor expressed on NK cells. SLAM family members are considered as important targets for anti-myeloma immunotherapy 50 . 2B4 positively regulates NK cell function and makes heterophilic interaction with CD48 51 . This complex is involved in Interleukin-2 associated propagation and activation of NK cells which leads to NK cell mediated cytotoxicity and tumour clearance 52 . The CC′ loop of the m2B4 comprising of residues 38 EQGSHR 43 (PDB:2PTT) 51 contributes 19.01% to the total 2B4 interaction-interface BSA. This CC′ loop is also seen to contribute two potential hydrogen bonds and one salt bridge (Fig. 2b). Furthermore, the CC′ loop of CD48 comprising of 37 TKNQK 41 (PDB:2PTT) contributes 28.92% to the total CD48 interaction-interface BSA, along with five potential hydrogen bonds (Fig. 2c). It appears that the observation of post-interaction movement of CC′ loop of 2B4 and CD48 is unique in this context (Fig. 2a). The CC′ loop of 2B4 is reported to be flexible in its apo form, among the four molecules in the asymmetric unit, this loop is not modelled in two molecules due to its disordered state. However, in the complex, the CC′ loop of 2B4 moves to accommodate CD48 and interacts with the 2B4, as mentioned earlier. This movement is characterized by the maximum deviation of about 4.2 Å of C α atom of H 42 , when apo (PDB:2PTU) and bound 2B4 (PDB:2PTT) were compared (Fig. 2a). As observed in the 2.4 Å structure, the CC′ loop of apo CD48 is highly flexible and was not modelled in any of the molecules in its asymmetric unit. However, upon complexation (as observed in 1.6 Å structure), the CC′ loop assumes a rigid hook like conformation and latches on to 2B4 (Fig. 2c). This is a unique feature where CC′ loops of both 2B4 and CD48 show post-interaction movements unlike FG loops of these proteins, which contribute 28.51% (2B4) and 30.89% (CD48) to the total interaction-interface BSA. Since, the CC′ loops of 2B4 and CD48 are located on opposite sides of each other, the post-interaction movement of both the CC′ loops seem to act like clamps to increase the binding affinity of the 2B4:CD48 interaction.
A similar post-interaction movement of the CC′ loop is seen in human PD-1 (hPD-1) when it interacts with human PD-L1 (hPD-L1) 53 . PD-1 is a well-studied inhibitory cell surface receptor 54 [55][56][57] . Its ligands PD-L1 and PD-L2 have been observed to be over-expressed on several kinds of tumour cells to escape immunosurveillance [58][59][60] . The CC′ loop of hPD-1 comprising residues 71 SPSNQ 75 (PDB:4ZQK) contributes 15.22% to the total PD-1 interaction-interface BSA and makes four hydrogen bonds with PD-L1. Q 75 of PD-1 is observed to contribute a BSA of 98.29Å 2 to the interaction-interface and makes two potential side-chain to main-chain hydrogen bonds with R 125 of PD-L1 and one side-chain to side-chain hydrogen bond with D 26 of PD-L1. Interestingly, a large conformational change is observed in the CC′ loop of PD-1 where a maximum deviation of 8.23 Å in C α atom of Q 75 is observed before and after its interaction with PD-L1 (Fig. 2d). In contrast, there is no post-interaction movement of the FG loop of PD-1 which contributes 17.41% of the total PD-1 interaction-interface BSA. Likewise, no post-interaction movement is seen in the CC′ loop and FG loop of its interacting partner PD-L1. Unlike 2B4:CD48, there is only one-sided clamping of the CC′ loop of PD-1. It is also interesting to note that, of all the residues comprising the CC′ loop, only Q 75 is completely conserved across most species (Fig. 2e). This opens the possibility to modify other residues of CC′ loop to enhance the binding affinity.

inducibly expressed on T-cells, NK cells, B-cells, dendritic cells and macrophages
Loop to β-strand transition of CC′ loop upon interaction. PD-L2 is another ligand which binds to PD-1 with three times higher affinity than PD-L1 61 . A unique feature in mPD-L2 is observed where, the part of CC′ loop of mPD-L2 undergoes a structural transition from a loop into a β strand conformation, upon binding with its partner mPD-1 (Fig. 3a). The CC′ loop of apo mPD-L2 comprises of residues 62 VENDTSLQSERA 73 (PDB: 3BOV) 62 , however upon binding to mPD-1, 67 SLQ 69 of the CC′ loop assumes a β strand conformation (PDB:3BP5) 62 (Fig. 3b). This could be attributed to the stabilization of the long CC′ loop of mPD-L2 after binding to mPD-1. The conformational change observed in the CC′ loop of PD-L2 is accompanied by a large movement of the CC′ loop of mPD-L2 towards mPD-1, upon binding to mPD-1, which is represented by a deviation of 9.5 Å of C α atom of D 65 of the CC′ loop of mPD-L2. However, for such a large post-interaction movement, the contribution of the CC′ loop to the total PD-L2 interaction-interface is quite low (7.38%) and we did not observe any polar interactions. A similar feature of post-interaction movements of CC′ loop and FG loop is observed in mPD-1, where a deviation of 3.2 Å in the C α atom of S 41 and a deviation of 5.62 Å in the C α atom of P 97 , was observed in CC′ loop and FG loop respectively (PDB:3BP5). The CC′ loop and FG loop of mPD-1 contributes 13.66% and 31.01% towards the interaction-interface BSA of mPD-1. The post-interaction movements of CC′ loops of mPD-1 and mPD-L2 and FG loop of mPD-1 causes a three-way clamping which might contribute to the increased binding affinity (Fig. 3c).

Unusual conformation of cc′ loop leads to non-canonical IgV domain.
The examples of ICR complexes involving IgV domains described in the previous sections are protein-protein complexes. There are a few examples of immune receptors having IgV domains that recognize biologically important small molecules, suggesting the versatility of this domain to carve its front face to recognize molecules as big as proteins and small molecules of the size of an amino acid. The relatively flat canonical front face of the IgV/Ig like domain does not seem to support a cleft that can accommodate binding of small molecules. Hence, for the IgV domain to acquire such a property, it appears that a non-canonical conformational change is required to accommodate small molecules (Fig. 4a). This non-canonical conformation change was observed for the first time in the case of murine T-cell immunoglobulin and mucin domain containing protein-3 (mTIM-3), where unusual conformation of the CC′ loop creates a cleft for small molecule binding 63 . mTIM-3 is primarily expressed on Th1 and dendritic cells 64 . www.nature.com/scientificreports www.nature.com/scientificreports/ They bring about apoptosis in Th1 cells via downstream inhibitory signalling, when phosphatidylserine (PtdSer) expressed on cells destined for apoptosis, bind to mTIM-3 65 . As depicted in the Fig. 4a,b, the CC′ loop of mTIM-3 is peeled out of the IgV domain core towards FG loop of mTIM-3, forming a narrow pocket between CC′ loop and FG loop, which is known to bind PtdSer. As explained in the introduction, in a canonical IgV/Ig domain, the tip of CC′ loop and FG loop are separated approximately by 25 Å and these two loops are located at the two ends of the IgV domain (Figs. 1b, 4a). This binding pocket carved between the CC′ and FG loops is conserved in all TIM family proteins 63,66,67 . Note that such unusual peeling of C and C′ strands as well as the connecting loop away from the core would fundamentally require (1) change in the side-chain functionality of the core forming hydrophobic residues which are exposed to the solvent due to this non-canonical conformation (2) a strong interaction that can peel the CC′ loop away from its canonical position to form a pocket with FG loop. As expected, this unusual conformation of CC′ loop is stabilized by two non-canonical disulphide bonds between C 33 of the C strand with C 44 of the CC′ loop and C 91 of F strand with C 39 of the CC′ loop (PDB:3KAA) (Fig. 4b) 68 . These non-canonical disulphide bonds pull the CC′ loop from its canonical position and away from the core towards the FG loop. Further, few of the core-forming residues of the C and C′ strands and those from the core that interact with these residues in canonical IgV domains, are changed to hydrophilic residues. This pocket is further stabilized by few hydrogen bonds and salt-bridges. The CC′ loop of mTIM-3 comprising of 55 KGFCPWSQCTN 65 (PDB:3KAA) contributes about 31.43% to the total mTIM-3 interaction-interface BSA and makes two potential hydrogen bonds with the serine moiety of PtdSer (Fig. 4b). W 41 , present on the tip of the CC′ loop acts as a gate for this pocket, regulating the entry of PtdSer. W 41 in the closed conformation connects the CC′ loop and FG loop via hydrophobic interactions (PDB:2OYP) 63 . However, in the presence of PtdSer, W 41 flips and rotates in such a manner that it moves away from the FG loop and allows PtdSer to enter the pocket (Fig. 4c). Mutagenesis studies suggested that mutation of 60 WSQ 62 to homologous human sequence of VFE critically reduced the binding affinity of mTIM-3 to PtdSer 68 . These observations and experiments emphasize the role of this non-canonical CC′ loop in forming the pocket for an IgV domain to interact with a small molecule. It also plays a role of gating the entry of the small molecule into the pocket.
Another important example of CC′ loop of the IgV domain assuming a non-canonical conformation to form a small molecule binding cleft is that of CD22. CD22 is a receptor present on the B-cells and maintains a basal level of inhibition of B-cell activation. CD22 is a member of the sialic acid-binding immunoglobulin-like lectins family (Siglecs), found only on B-cells 69 and binds specifically to α2-6 siallylactose. Since, CD22 is purported to bind to α2-6 siallylactose, a non-canonical feature is observed similar to that of mTIM-3, where the C, C′ strands and CC′ loop move away from the core to form a unique pocket (PDB:5VKM) 70 . The C and C′ strands of CD22 are bent towards the N-terminal forming a β-hairpin. This β-hairpin structure along with the FG loop forms a comparatively wide but shallow pocket, where α2-6 siallylactose binds to CD22 (Fig. 4d). www.nature.com/scientificreports www.nature.com/scientificreports/ As mentioned earlier, the non-canonical conformation of the CC′ loop to form a pocket with FG loop could be due to the presence of hydrophobic residues like I 57 , F 59 , P 62 and Y 64 on the C strand and F 71 on C′ strand in CD22 which forms a hydrophobic core on the front face (Fig. 4d). This hydrophobic core enables the C and C′ strand to move towards the FG loop. Furthermore, the presence of polar residues like N 61 , E 63 and N 65 on the C strand and D 72 and T 74 on the C′ strand, which would be facing the hydrophobic core of a canonical IgV domain, might have pushed the C and C′ strand away from the protein hydrophobic core due to steric clashes.
In a recent study that describes the structure of CD160:HVEM complex (PDB:6NG3) 71 , it was observed that a similar reorientation of C and C′ strands together with a single non-canonical disulphide bridge creates a pocket in the front-face of CD160 ( Supplementary Fig. S2), which is wider than that is observed in mTIM-3 and CD22. The dimension of the HVEM being slightly smaller than the IgV domains, and obviously wider than the small molecules, requires complementary surface on CD160 that would exactly fit the dimensions of the HVEM. It is interesting to note that unlike TIM family members with two disulphide bridges on the front-face, how a relative wider surface is created using single disulphide bridge. Furthermore, point mutations on the CC′ loop of CD160, resulted in the reduced binding to HVEM. All these results suggest that nature uses the versatility of C and C′ strands together with CC′ loop to create ligand binding surface of different dimensions.
A question piqued our interest in these structures, why does the CC′ loop assume a non-canonical conformation to form a pocket? Why doesn't the onus fall on FG loop to form this non-canonical conformation? Upon closer observation, it might be due to the presence of the canonical disulphide bond between the B strand and F strand of the IgV domain. This covalent bond might restrict the flexible movement of the FG loop and/or F strand. Hence, we propose that, since CC′ loop is not constrained by the presence of any covalent bonds or other www.nature.com/scientificreports www.nature.com/scientificreports/ rigidity conferring interactions, it is a prime candidate in the front face to assume non-canonical conformations to incorporate and provide additional affinity to its respective binding partner.
Along with this analysis, existing mutagenesis studies on various ICRs revealed that CC′ loop made key interactions with their binding partners (Supplementary Table 1). Here, of the 11 mutagenesis studies, nine of them revealed that mutations on CC′ loop resulted in either disruption of interaction or reduction in affinity. These mutations were selected purely based on their location at the receptor:ligand interface as observed in the complex structures without any importance to CC′ loop. It should also be noted that a mutagenesis study on CC′ loop, where CC′ loop of Nectin-2 was replaced with that of PVR showed increased affinity to TIGIT 45 , closely mimicking the higher affinity observed between TIGIT and PVR compared to TIGIT and Nectin-2. This result suggests that the rational modification of CC′ loop might help to produce ICRs with desired affinity.

Modification of CC′ loop might lead to increase in binding affinity: A case study of TIGIT/Nectin
pathway. T-cell immunoreceptor with Ig and ITIM domain (TIGIT) is an inhibitory receptor expressed on NK cells and certain subset of T-cells. TIGIT belongs to the PVR/Nectin family 72 and is responsible for blocking the anti-tumor activity in NK cells 73 and T-cells 72 . TIGIT binds to itself, PVR and Nectin-2 in a canonical mode where the Y/FP motif present on the FG loop of all the three proteins, recognizes a structurally conserved complementary cleft on the front face of their partners 38,45,46 . Superposition of human TIGIT:TIGIT, TIGIT:Nectin-2 and TIGIT:PVR structures, clearly suggests that TIGIT binds to all its binding partners and to itself in the same manner due to the 'lock and key' mode of interaction, but with differing binding affinity 38 . Among a few factors for these varied binding affinities between TIGIT and its ligands, the differing lengths and side-chain functionalities of the residues of CC′ loops of TIGIT, Nectin-2 and PVR (Fig. 5a) was proposed as a key factor 38,45 . This similarity in the mode of association of TIGIT with itself and with Nectin-2 and PVR gives us an opportunity to computationally investigate the impact of the CC′ loop on the binding affinity. Considering these observations, TIGIT/ Nectin pathway presents itself as a prime example for investigating such a hypothesis. The CC′ loop of TIGIT is the shortest with 4 polar residues ( 61 QQDQ 64 ) (PDB:3RQ3), Nectin-2 has the longest CC′ loop with 9 residues ( 72 RPDAPANHQ 80 ) (PDB:5V52) 45 and PVR has a 7-residue long CC′ loop ( 69 HGESGSM 75 ) (PDB:3UDW) 46 . The CC′ loop of TIGIT makes no contribution to the interaction-interface of the TIGIT homodimer, very weak Van der Waals interactions are provided by the residues of CC′ loop of Nectin-2 towards the interaction-interface of TIGIT:Nectin-2 heterodimer. However, a mix of Van der Waals and H-bond interactions are provided by the CC′ loop of PVR towards the interaction-interface of TIGIT:PVR heterodimer (Table 1) (Fig. 5b-d). This concept is well elucidated by the experimental swapping of CC′ loop of Nectin-2 with the CC′ loop of PVR. This particular Nectin-2 mutant displayed a higher affinity towards TIGIT than wild type Nectin-2 45 . In line with this mutagenesis study, we tried to apply a similar concept of CC′ loop swapping on TIGIT monomer. Since, TIGIT forms a weak homodimer and the contribution from its CC′ loop is low, could the binding affinity of TIGIT with itself be increased by splicing its CC′ loop with that of the CC′ loops of PVR and Nectin-2 on one of the protomers of the TIGIT homodimer? Experimentally determined structures of TIGIT:PVR (PDB:3UDW) and TIGIT:Nectin-2 (PDB:5V52) and TIGIT:TIGIT homodimer (PDB:3RQ3) were superposed in the crystallographic modelling program Coot. In two independent attempts, CC′ loops of one TIGIT monomer of the TIGIT homodimer (PDB:3RQ3) was swapped with the CC′ loop of Nectin-2 and PVR.
The complex structure thus created comprising of TIGIT with the CC′ loop of Nectin-2 (named T N ) and wild type TIGIT (WT-T) was subjected to energy minimization using UCSF Chimera with Amber ff14SB forcefield. The same was done with the heterodimer containing TIGIT with the CC′ loop of PVR (T P ) and WT-T. Since, the mode of binding of TIGIT:TIGIT homodimer and TIGIT:PVR/Nectin-2 heterodimers are similar, the resulting energy minimized structures of WT-T:WT-T, T P :WT-T and T N :WT-T were docked using RosettaDock via ROSIE server.
To examine the veracity of the docking algorithm, docking trials were also performed on experimentally determined TIGIT:TIGIT, TIGIT:Nectin-2 and TIGIT:PVR complexes. It was observed that the interface score was a more accurate parameter than the docking score, when the scores were compared with the docked poses which were in accordance (RMSD < 1) with the experimentally determined structures. Hence, these interface scores (more negative score, better the strength of interaction) were used as a reference in order to compare with that of the modified heterodimers (T P :WT-T and T N :WT-T). Results showed that the WT-T:WT-T had an interface score of −8.2. The best docked poses of T N :WT-T and T P :WT-T gave interface scores of −8.4 and −9.1 respectively (Fig. 5e,f respectively). It is interesting to note that difference in the interface scores of T N :WT-T and WT-T:WT-T (−0.2) is higher than that between T P :WT-T and WT-T:WT-T (−0.9), which indicates that the contribution of the CC′ loop of PVR plays an important role in the interaction-interface than the CC′ loops of TIGIT and Nectin-2. It can be expected that the differences in interaction due to other front face residues of Nectin-2 and PVR also contribute to the observed differences in affinity between TIGIT:Nectin-2 and TIGIT:PVR complexes. However, the docked T N :WT-T and T P :WT-T structures are similar to the experimentally determined (PDB:3UDW and 5V52 respectively) structures and maintain similar interactions seen between the CC′ loops of PVR and Nectin-2 with the WT-T. The interface scores are reflective of the fact that CC′ loop of Nectin-2 makes very few hydrophobic interactions, whereas CC′ loop of PVR makes significant amount of hydrophobic and hydrophilic interactions, which is also observed in the experimentally determined structures. This increase in the interaction score of T P :WT-T is due to the additional interactions made by the CC′ loop of PVR with the cognate TIGIT monomer, similar to what is seen in TIGIT:PVR. Conversely, it was observed that when the CC′ loop of PVR was replaced by the CC′ loop of TIGIT (P T ) and docked with the WT-T in the canonical orientation, there were loss of important interactions which is also supported by interface score of −6.4. These interface scores indicate that the modifed versions of TIGIT (T P and T N ) have a higher binding affinity with its corresponding monomer of TIGIT (WT-T) than wild-type TIGIT itself. Interestingly, mere increase in the length of the CC′ loop doesn't translate into increased binding affinity as the length of CC′ loop of PVR is shorter than that of Nectin-2.
Functionalities of the residues present in the CC′ loop and their interactions with the cognate ligand appear to take precedence. These results might provide the motivation to proceed with wet lab experiments to confirm our hypothesis with not only TIGIT/Nectin pathway, but other medically relevant ICRs as well.

conclusion
The immune checkpoint blockade has brought about a paradigm shift in cancer therapy, however, rising reports of toxicity of mAbs coupled with its high cost, suspected resistance, requirement of high dose, poor solid tumour penetration and so on, have pushed for alternatives that can mimic the function of mAbs. Since, the soluble isoforms of these ICRs are naturally used by our immune system to tweak the immune response, rationally designed naturally occurring ICRs to mimic the function of therapeutic mAbs appears to be an effective approach. However, to design such a protein, sound structural information of the proteins and intuitive modification strategies are critical. In this study, the CC′ loop appears to influence the binding affinity of IgV domain containing ICRs in about half the ICR complexes analysed. Further, in about quarter of these complexes, the CC′ loop contributes more towards the interaction-interface than the FG loop. The CC′ loop is flexible enough to accommodate the incoming ligand and once bound, it appears to act like a clamp to increase the binding affinity of the complex. The CC′ loop does not arbitrarily move to accommodate the incoming ligand. Its movement is specific for a specific ligand, as clearly elucidated with the movement of CC′ loop of PVR, when bound to TIGIT (moves towards) and CD96 (moves away) 48 . In a few cases, the CC′ loop takes a preformed non-canonical conformation, as observed in the case of TIM-3 and CD22. This work attempts to provide novel insights for the rational design of soluble ICRs to mimic the role essayed by mAbs with an expected low toxicity and cost and suggests CC′ loop of the IgV domains of ICRs is the probable hot-spot for modification. Structure and interaction analysis. The interactions amongst the ICRs and their ligands were analysed using PDBe PISA 76 , where H-bond length not greater than 3.5 Å and not smaller than 2.5 Å were considered. Van der Waals interactions were considered if the two residues were separated by a distance of less than 5 Å. Coot 77 and PyMol 78 were used to visualize the complexes and their interactions. Buried surface area (BSA) of each monomer in the complex was considered for calculating the contribution of CC′ loop of the respective monomer which is indicative of Van der Waals interactions. The post-interaction movement of CC′ loop was considered significant, if the maximum deviation of the C α atom of any residue which belongs to CC′ loop is more than 3 Å. The sequence logos highlighting the conservation of the residues of CC′ loop have been performed using WebLogo 79 . In silico modelling and energy minimization trials. In silico splicing of the CC′ loops of PVR and Nectin-2 onto one TIGIT monomer of the TIGIT homodimer was performed using Coot using experimentally solved structure of TIGIT homodimer in hexagonal space group (PDB: 3RQ3) as the template structure. The CC′ loops of PVR and Nectin-2 were excised and replaced the CC′ loop of TIGIT by superposing TIGIT homodimer structure with TIGIT:PVR (PDB:3UDW) and TIGIT:Nectin-2 (PDB:5V52) heterodimers independently. These in silico modified heterodimers of TIGIT (wild type TIGIT with TIGIT with CC′ loop of Nectin-2 or PVR) were subjected to energy minimization using UCSF Chimera's Amber ff14SB forcefield 80 . These values were documented for further analysis.
Protein-protein docking trials. As mentioned earlier, the mode of interaction of TIGIT homodimer and heterodimers are the same. Hence, the modified TIGIT heterodimers were docked using RosettaDock 81,82 via ROSIE server 83 . Since, the modified heterodimers were derived from the exact pose of the experimentally determined structure (PDB:3RQ3), interface score was given paramount importance. The interface score was found to be more relevant when the docked poses were compared with experimentally determined structures. This parameter would help in inferring the trend associated with the impact of modification of the CC′ loop. The docked pose with the highest interface score which had a RMSD <1 with the submitted template structure, was considered for further investigations. Negative interface score indicates stronger interactions between the interfaces of the two proteins. Furthermore, thorough structural analysis was carried out on the selected docked pose to ascertain the number and type of interactions made by the CC′ loop with its binding partner. These interactions were compared with the interactions made by the CC′ loops of PVR and Nectin-2 with TIGIT as seen in experimentally determined structures (PDB:3UDW and 5V52 respectively) using PDBe PISA 76 and PIC 84 .

Data availability
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