**4. Optimized selection of patients/donors**

#### **4.1. Clinical outcomes: on the KIR ligand mismatch model**

Research reports focusing on the KIR of NK cells and the HLA of tumor cells for the purpose of treating leukemia have been drawing attention since the 2000′s. In the report by Ruggeri et al. published in *Science* in 2002, the cases in which HLA‐ABC recognizable by NK cells is present in a donor but not in a recipient were defined as "KIR mismatch". It was speculated that in such cases, NK cells not receiving suppression signals from the donor HLA attack the cells of the recipient. When Ruggeri et al. started this research, it was not known that NK cells are a het‐ erogeneous population expressing multiple inhibitory receptors, or that the specific antibodies that can be used for analysis are inadequate. Ruggeri and colleagues, therefore, examined only the HLA of donors and recipients without examining the KIR, and they analyzed patients divided into the two groups of HLA matched and mismatched transplantations. Although it may not have been an appropriate observation based on the current knowledge, at that time, the analysis was based on the following basic research data.

similar to that of FcγRIIa), suggesting that it transmits a signal via SRC‐SYK (the SRC family of kinases and spleen tyrosine kinase [SYK]) signaling pathways [95]. However, the expres‐ sion of CD32c was less than half of that of NK cells [96, 97]. Many studies focusing on CD16

Several genetic polymorphisms of CD16 exist. Among them, the amino acid at position 158 has been shown to be important for the strength of the affinity for antibodies. The affinity depends on whether the amino acid at position 158 is phenylalanine or valine, and the valine type (158 V) has a higher affinity for the Fc of IgG. A number of reports have indicated that differences in the affinity for antibodies are correlated with therapeutic effects, and many studies have analyzed the clinical responsiveness of this gene polymorphism and antibody therapy. Cartron et al. examined the effects of rituximab treatment for non‐Hodgkin's lym‐ phoma, and they reported a higher objective response rate in CD16 (158 V) homozygous patients compared to CD16 (158F) carrier patients [98]. Wang et al. analyzed the outcomes of rituximab treatment for Follicular Lymphoma and reported significantly more likely progres‐ sion‐free survival at 2 years in CD16 (158 V) homozygous patients compared to CD16 (158F)

These results suggest that the affinity of CD16 for antibodies correlates with the therapeutic effect. Thus, focusing on CD16, the modification of NK cells has been attempted. Binyamin et al. reported that introducing CD16 (158 V) into NK‐92 cells not expressing CD16 improved the cytotoxicity against B‐cell lymphoma with rituximab [100]. Carlsten et al. reported high ADCC activity of cultured NK cells from healthy donors with CD16 (158F/F) and transduced CD16

As described above, since NK cells mediate antibody‐dependent cytotoxic activity via Fc receptors, compatibility with antibody drugs targeting ADCC is desirable. As a strategy to further augment the antitumor effect, a plausible strategy is to enhance the affinity between the Fc receptor and the antibody. Low‐molecular‐weight compounds that are able to inhibit the shedding of CD16 have been reported. It is known that CD16 is cleaved by a protease such as a disintegrin and a metalloprotease 17 (ADAM17) when cells are activated [102], and thus in order to exert more sustained and enhanced ADCC activity, a method of inhibiting the cleavage of CD16 on NK cells may be important. In fact, inhibitors of ADAM17 enhanced the activity of NK cells [102, 103]. Another method to inhibit the cleavage of CD16 is a genetic modification. The substitution of the serine residue at position 197 in CD16 by a proline pre‐ vents the cleavage of CD16 on NK cells [104]. It may be possible to promote the antitumor

Research reports focusing on the KIR of NK cells and the HLA of tumor cells for the purpose of treating leukemia have been drawing attention since the 2000′s. In the report by Ruggeri et al.

B‐cell lymphoma cells [101].

have thus been conducted.

98 Natural Killer Cells

carrier patients, at 45 and 14%, respectively [99].

effect by using a CD16 mutant.

**4. Optimized selection of patients/donors**

**4.1. Clinical outcomes: on the KIR ligand mismatch model**

(158 V) mRNA by electroporation against rituximab‐coated CD20<sup>+</sup>

Ruggeri et al. examined whether CD56‐positive NK cell clones collected from donors could attack leukemia cells of the recipient. They found that NK cell clones that attack leukemia cells exist, and their high frequency (≥2%) correlates well with the cases in which the donor's and recipient's KIR ligand (HLA) do not match in the GVH direction. Ruggeri et al. analyzed 35 acute lymphoblastic leukemia (ALL) and 57 acute myeloid leukemia (AML) patients who had allogeneic hematopoietic stem cell (HSC) transplantations at their institution. The result was a breakthrough result in 20 AML patients who were not KIR ligand mismatches in the GVH direction, with 0% relapse after transplantation [105]. This report was the subject of much attention, and then it was decided to conduct data analyses in medical institutions around the world seeking reproducibility of the results.

The analysis by Ruggeri et al. was by the method known as the "KIR ligand mismatch model", which examines only the donor and recipient HLA‐ABC without checking the KIR. This method determines whether a KIR match or mismatch is determined. Researchers all over the world could thus easily use this method. However, most of the analysis results obtained in this way conflicted with the report by Ruggeri et al.

As an example, we will describe the analysis results obtained from the study of the Japan Marrow Donor Program (JMDP). In 2007, Morishima et al. reported the results of their analy‐ sis of 1790 patients who underwent an allogeneic bone marrow transplantation in accord with the KIR ligand mismatch model [106]. In conventional domestic allogeneic HSC transplanta‐ tion, the criterion for donor selection is that the HLA‐AB and ‐DR matched, and by this crite‐ rion 534 of the 1790 cases were HLA‐C mismatched. As a result, the overall survival rate, the recurrence rate, and the incidence of GvHD were poor in the group in which the KIR ligand HLA‐C was mismatched in the GVH direction.

The finding that allogeneic HSC transplantation has no merit in KIR mismatches is common in the report about cases from the U.S. National Marrow Donor Program (NMDP) and the European group for blood and marrow transplantation (EBMT) [107]. Comparing Ruggeri's 2002 report with Morishima's 2007 report, the biggest difference was the cell source for trans‐ plantations, the former being CD34+ cell transplantation from haploidentical donors and the latter being conventional bone marrow transplantation. In addition, Ruggeri et al. used anti‐ thymocyte globulin (ATG) in all cases. A re‐analysis of the registered JMDP cases reconfirmed that the transplantation performance of the HLA‐C matched group was also good and that of the group with the HLA‐C mismatch was poor [108].

Interestingly, the disadvantage of this HLA‐C mismatch was not observed in the ATG admin‐ istration group. In other words, it is suggested that not NK cell dysfunction but T cells induced by HLA‐C mismatch had exacerbated the transplantation results. T cell‐depleted grafting and ATG might avoid the T‐cell response. There are certain reports that HLA‐C mismatches are recognized by cytotoxic T lymphocytes (CTLs). There are two papers that reported a total of nine CTL clones from two patients who developed GvHD [109, 110]. Interestingly, all of the targets of the nine CTL clones were HLA‐C, which was different from the HLA‐C of the recipient. There is no doubt that the difference in HLA‐C could be a target for CTL.

#### **4.2. Receptor ligand model or missing ligand model**

Although the KIR ligand mismatch model investigated only ligand (i.e., HLA) differences, eventually some researchers noted that donors' KIR should also be examined and analysed by different approaches were developed. In 2004, Leung et al. analyzed 36 children who received selective CD34+ cell transplantation from haploidentical donors [111]. They first examined the presence or absence of inhibitory KIR in donor cells by flow cytometry (i.e., a phenotype assay). It was speculated that the recipient would have a KIR mismatch if the recipient did not have a ligand for KIR (suppressing type) of the donor. This is the "recep‐ tor‐ligand model".

For example, when the donor has KIR2DL1 with HLA‐C1/C1, when the recipient is HLA‐ C1/C1, it is considered a KIR match in the KIR ligand mismatch model, but it is a ligand mismatch of KIR2DL1 in the receptor‐ligand model. This is a very frequent combination for Japanese (HLA‐C1/C1 = 85%, KIR2DL1<sup>+</sup> = 99%). Conversely, if the donor does not have KIR2DL1 with HLA‐C1/C2, if the recipient is HLA‐C1/C1, it is considered a KIR mismatch in the KIR ligand mismatch model, but it is considered a KIR match in the receptor‐ligand model. However, since there are few KIR2DL1<sup>−</sup> and HLA‐C1/C2 among Japanese, this com‐ bination is extremely rare.

An analysis conducted to determine which model can predict recurrence more accurately revealed that the receptor‐ligand model is superior [111]. Hsu et al. also reported their analy‐ sis based on the receptor‐ligand model [112]. They investigated 178 cases of T cell‐depleted transplantation and found that the positive rates of KIR2DL1, KIR2DL2/3, and KIR3DL1 were 93, 99, and 92%, respectively, in the donor gene, and that although there were 112 cases (63%) of the 178 patients who were expected to exhibit the GVL effect, there was a significant dif‐ ference in the recurrence rate, and the disease‐free survival rate/overall survival rate was also good in the AML and myelodysplastic syndrome (MDS) patients.

Hsu et al. presented a new idea in 2006 [113]. It is hard to examine the donor's KIR by genetic testing, but donors usually have genes of KIR2DL1, KIR2DL2/3, and KIR3DL1. Therefore, HLA‐C1/C1 homozygous, HLA‐C2/C2 homo and HLA‐Bw6/Bw6 homo patients identified by examining only the recipient's HLA may experience the GVL effect from donor NK cells. This method is called the "missing ligand model". It was designed for transplantation perfor‐ mance analyses, not for donor selection. Hsu et al. analyzed 1770 patients who had allogeneic (unrelated) T‐cell‐depleted transplantation. A University of Minnesota study examined 2062 cases from the U.S. NMDP [114], and based on the idea that NK cells have a GVL effect, they excluded 568 Japanese subjects or analyzed only partial diseases with good prognosis. Nonetheless, that paper is very interesting as it shows the difference in the distribution of KIR ligand between Japanese and Westerners and the difference in the recurrence rate of disease [113]. We can see how Japanese are 'biased' toward HLA‐C1.

Interestingly, the disadvantage of this HLA‐C mismatch was not observed in the ATG admin‐ istration group. In other words, it is suggested that not NK cell dysfunction but T cells induced by HLA‐C mismatch had exacerbated the transplantation results. T cell‐depleted grafting and ATG might avoid the T‐cell response. There are certain reports that HLA‐C mismatches are recognized by cytotoxic T lymphocytes (CTLs). There are two papers that reported a total of nine CTL clones from two patients who developed GvHD [109, 110]. Interestingly, all of the targets of the nine CTL clones were HLA‐C, which was different from the HLA‐C of the

Although the KIR ligand mismatch model investigated only ligand (i.e., HLA) differences, eventually some researchers noted that donors' KIR should also be examined and analysed by different approaches were developed. In 2004, Leung et al. analyzed 36 children who

examined the presence or absence of inhibitory KIR in donor cells by flow cytometry (i.e., a phenotype assay). It was speculated that the recipient would have a KIR mismatch if the recipient did not have a ligand for KIR (suppressing type) of the donor. This is the "recep‐

For example, when the donor has KIR2DL1 with HLA‐C1/C1, when the recipient is HLA‐ C1/C1, it is considered a KIR match in the KIR ligand mismatch model, but it is a ligand mismatch of KIR2DL1 in the receptor‐ligand model. This is a very frequent combination

KIR2DL1 with HLA‐C1/C2, if the recipient is HLA‐C1/C1, it is considered a KIR mismatch in the KIR ligand mismatch model, but it is considered a KIR match in the receptor‐ligand

An analysis conducted to determine which model can predict recurrence more accurately revealed that the receptor‐ligand model is superior [111]. Hsu et al. also reported their analy‐ sis based on the receptor‐ligand model [112]. They investigated 178 cases of T cell‐depleted transplantation and found that the positive rates of KIR2DL1, KIR2DL2/3, and KIR3DL1 were 93, 99, and 92%, respectively, in the donor gene, and that although there were 112 cases (63%) of the 178 patients who were expected to exhibit the GVL effect, there was a significant dif‐ ference in the recurrence rate, and the disease‐free survival rate/overall survival rate was also

Hsu et al. presented a new idea in 2006 [113]. It is hard to examine the donor's KIR by genetic testing, but donors usually have genes of KIR2DL1, KIR2DL2/3, and KIR3DL1. Therefore, HLA‐C1/C1 homozygous, HLA‐C2/C2 homo and HLA‐Bw6/Bw6 homo patients identified by examining only the recipient's HLA may experience the GVL effect from donor NK cells. This method is called the "missing ligand model". It was designed for transplantation perfor‐ mance analyses, not for donor selection. Hsu et al. analyzed 1770 patients who had allogeneic (unrelated) T‐cell‐depleted transplantation. A University of Minnesota study examined 2062

cell transplantation from haploidentical donors [111]. They first

= 99%). Conversely, if the donor does not have

and HLA‐C1/C2 among Japanese, this com‐

recipient. There is no doubt that the difference in HLA‐C could be a target for CTL.

**4.2. Receptor ligand model or missing ligand model**

for Japanese (HLA‐C1/C1 = 85%, KIR2DL1<sup>+</sup>

model. However, since there are few KIR2DL1<sup>−</sup>

good in the AML and myelodysplastic syndrome (MDS) patients.

received selective CD34+

bination is extremely rare.

tor‐ligand model".

100 Natural Killer Cells

Although few cases of GvHD in Japanese have been reported, the recurrence rate of HLA‐C1/ C1 patients is not much different from that of Westerners. In addition, among the Japanese, the recurrence rate is extremely small when the recipients are HLA‐C2/C2 homozygous, which is a minority. However, caution is required in the interpretation of the results, as there are only three cases in which the recipient had HLA‐C2/C2. Further detailed analysis is expected in the future.

#### **4.3. Therapeutic effects of allogeneic transplantation of NK cells, and their limitations**

In 2007, Ruggeri et al. reported the results of 122 cases of AML [115], which included 57 cases [105] and the other 55 cases. In the KIR ligand mismatch model, there were 51 cases of KIR mismatch in the GVH direction and 61 cases of matches, and the number of remission cases at the time of transplantation and the cases which were refractory to treatment were approxi‐ mately 50% of the cases. The results showed that although the relapse rate was significantly lower in the cases of remission at the time of transplantation in the KIR mismatch in the GVH direction (3 vs. 47%, p = 0.003), in the cases in which the treatment was refractory, no treat‐ ment effect was observed (32 vs. 37%, p = NS).

Is it true that NK cells exert a GVL effect? A direct answer to that question has been reported as a KIR‐mismatch NK cell transplantation in recent years. Ten children with AML [7] and 13 adults with AML [116] underwent the transplantation of CD56‐positive NK cells harvested from haploidentical donors. The collected NK cells were considered KIR mismatches, and in both studies, only NK cells were transplanted after pretreatment (fludarabine and cyclo‐ phosphamide). The number of transplanted NK cells was 29 × 10<sup>6</sup> /kg for the children and 2.7 × 10<sup>6</sup> /kg for the adults; the numbers of transplanted T cells were <1 × 10<sup>3</sup> /kg and < 1 × 105 /kg, respectively. After transplantation, 1–10 million units of IL‐2 were administered every other day, 6 times.

As a result, transient engraftment of donor NK cells was observed in all cases. Donor‐derived NK cells occupied 7% (1–30%) of the peripheral blood lymphocytes at the peak of day 14 in the children's report, and at day 28, donor‐derived NK cells have been detected from three of 10 patients [7]. All of the child patients had AML in complete remission, and all cases did not recur. Among the adult patients, whose leukemia was worse, one of five patients with clear recurrence showed transient remission, and two patients with genetic recurrence AML were remitted [116]. From the above results, it was demonstrated that NK cells exert a sufficient antitumor effect if the residual tumor is relatively small. However, it is not yet clear whether the therapeutic effect is proportional to the number of NK cells administered. It should be noted that even though transplanted CD3‐positive cells are limited to ≤1 × 10<sup>3</sup> /kg, there was no case of GvHD onset in either group.

#### **4.4. Selection of KIR in allogeneic hematopoietic stem cell transplant donors**

The target diseases in which the therapeutic effect by NK cells is confirmed in analyses of vari‐ ous clinical tests are mostly limited to AML. Clinical trials using conventional NK cells often do not even target other diseases. There are reports that NK cells do not show a therapeutic effect against ALL because of lymphoid cells highly MHC class I, and therefore, the inhibi‐ tory signal is strong [117]. In addition, KIR ligand mismatch can lead to GvHD by CTLs as described above. In order to avoid this, ingenuity such as umbilical cord blood transplanta‐ tion or the use of ATG may be necessary.

#### *4.4.1. Donor selection based on inhibitory receptors*

There is a reason to expect the GVL effect in donor NK cells not receiving a KIR signal from the recipient HLA. The main inhibitory receptors are KIR2DL1, KIR2DL2/3, KIR3DL1, and KIR3DL2. There is no evidence that the effects of these four inhibitory receptors are identical. Particularly with regard to KIR3DL2, there are reports that the KIR‐positive NK cells are not even licensed, even if the recipient has HLA‐A3 or A11 (KIR3DL2 ligands) [118]. Most NK cell transplantations have been expected to provide a therapeutic effect due to missing self of KIR2DL1 or KIR2DL2/3, and the number of transplantations in which a therapeutic effect by missing self of KIR3DL1 have been expected is very low. There is no transplantation in which the therapeutic effect by missing self of KIR3DL2 alone is expected.

For example, if the recipient is HLA‐C1/C1 if the KIR ligand mismatch model is used, an HLA‐C1/C2 or HLA‐C2/C2 donor would be selected. Even if transplantation is assessed using the receptor‐ligand model, the donor's KIR is often unknown. However, in Japanese, most (approx. 99%) donors KIR2DL1 can be considered positive. If so, in the receptor‐ligand model, it seems that any donor could be chosen, but NK cells derived from an HLA‐C1/C1 donor may show a lower GVL effect. When the recipient is HLA‐Bw6/Bw6, it must first be confirmed that all of the HLA‐A23, ‐24 and ‐32 are negative, and then an HLA‐Bw4‐positive donor should be selected. However, since 7% of Japanese are negative for KIR3DL1 gene, it must be confirmed that KIR3DL1 is positive for the donor by flow cytometry, or that the genotype is either KIR3DL1\*01502 or KIR3DL1\*020.

#### *4.4.2. Donor selection based on haplotypes*

It was reported that donors should be chosen for haplotype B when considering the activat‐ ing receptors [34]. In that study, Cooley et al. analyzed 448 AML cases of NMDP, and they observed that the 3‐year overall survival rate when the donor was haplotype BX was 31%, significantly higher than the rate of 20% when it was haplotype AA (p < 0.01). In addition, the recurrence decreases the most in cases that the donor had Cen‐B/Cen‐B, and it is reported that Tel‐B also leads to a reduction of recurrence and improvement of prognosis [119].

Based on the data from three groups in the United States (Memphis, Sloan Kettering, Minnesota), Leung et al. advocated a donor selection algorithm for NK cells [120]; in the trans‐ plantation of T cells containing bone marrow or peripheral blood stem cells, donors should be HLA matches, and donors with KIR ligand mismatch should be avoided. Conversely, in T cell‐depleted transplantation or umbilical cord blood transplantation, a donor for a KIR ligand mismatch should be chosen. It is also recommended that a donor with KIR that can attack the recipient's HLA (possibly KIR haplotype B) be selected. In Japan as well, if KIR hap‐ lotype testing of the donor banks becomes possible in the future, or if the NK cell preparation is put to clinical use, it will be possible to test this algorithm described by Leung et al.

#### *4.4.3. Donor selection based on activating receptor*

**4.4. Selection of KIR in allogeneic hematopoietic stem cell transplant donors**

tion or the use of ATG may be necessary.

102 Natural Killer Cells

*4.4.1. Donor selection based on inhibitory receptors*

the therapeutic effect by missing self of KIR3DL2 alone is expected.

genotype is either KIR3DL1\*01502 or KIR3DL1\*020.

*4.4.2. Donor selection based on haplotypes*

The target diseases in which the therapeutic effect by NK cells is confirmed in analyses of vari‐ ous clinical tests are mostly limited to AML. Clinical trials using conventional NK cells often do not even target other diseases. There are reports that NK cells do not show a therapeutic effect against ALL because of lymphoid cells highly MHC class I, and therefore, the inhibi‐ tory signal is strong [117]. In addition, KIR ligand mismatch can lead to GvHD by CTLs as described above. In order to avoid this, ingenuity such as umbilical cord blood transplanta‐

There is a reason to expect the GVL effect in donor NK cells not receiving a KIR signal from the recipient HLA. The main inhibitory receptors are KIR2DL1, KIR2DL2/3, KIR3DL1, and KIR3DL2. There is no evidence that the effects of these four inhibitory receptors are identical. Particularly with regard to KIR3DL2, there are reports that the KIR‐positive NK cells are not even licensed, even if the recipient has HLA‐A3 or A11 (KIR3DL2 ligands) [118]. Most NK cell transplantations have been expected to provide a therapeutic effect due to missing self of KIR2DL1 or KIR2DL2/3, and the number of transplantations in which a therapeutic effect by missing self of KIR3DL1 have been expected is very low. There is no transplantation in which

For example, if the recipient is HLA‐C1/C1 if the KIR ligand mismatch model is used, an HLA‐C1/C2 or HLA‐C2/C2 donor would be selected. Even if transplantation is assessed using the receptor‐ligand model, the donor's KIR is often unknown. However, in Japanese, most (approx. 99%) donors KIR2DL1 can be considered positive. If so, in the receptor‐ligand model, it seems that any donor could be chosen, but NK cells derived from an HLA‐C1/C1 donor may show a lower GVL effect. When the recipient is HLA‐Bw6/Bw6, it must first be confirmed that all of the HLA‐A23, ‐24 and ‐32 are negative, and then an HLA‐Bw4‐positive donor should be selected. However, since 7% of Japanese are negative for KIR3DL1 gene, it must be confirmed that KIR3DL1 is positive for the donor by flow cytometry, or that the

It was reported that donors should be chosen for haplotype B when considering the activat‐ ing receptors [34]. In that study, Cooley et al. analyzed 448 AML cases of NMDP, and they observed that the 3‐year overall survival rate when the donor was haplotype BX was 31%, significantly higher than the rate of 20% when it was haplotype AA (p < 0.01). In addition, the recurrence decreases the most in cases that the donor had Cen‐B/Cen‐B, and it is reported that

Based on the data from three groups in the United States (Memphis, Sloan Kettering, Minnesota), Leung et al. advocated a donor selection algorithm for NK cells [120]; in the trans‐ plantation of T cells containing bone marrow or peripheral blood stem cells, donors should be HLA matches, and donors with KIR ligand mismatch should be avoided. Conversely, in

Tel‐B also leads to a reduction of recurrence and improvement of prognosis [119].

KIR2DS1 is activated by HLA‐C2. This means that if the recipient is positive for HLA‐C2 and the donor is KIR2DS1‐positive (Tel‐B = haplotype BX), KIR2DS1‐positive NK cells will be activated and will kill the recipient's tumor cells. In actual transplantation, when NK cells were cultured *in vitro* to examine the antitumor effect on leukemic cells of patients, KIR2DS1‐ positive NK cells killed tumor cells of recipients with HLA‐C2 [121]. An analysis of the KIR gene of AML donors and recipients (1277 cases) from the U. S. NMDP and the Center for International blood and marrow transplant research (CIBMTR) provided large‐scale proof using actual transplantations, and the report was published in *The New England Journal of Medicine* [122]. The analysis revealed that recurrence was significantly reduced when the donor had KIR2DS1 (26.5 vs. 32.5% (KIR2DS1‐negative), p = 0.02). However, this effect was canceled when the donor was HLA‐C2/C2. This is thought to be a disarming phenomenon, and it can be explained as follows: the HLA‐C2/C2‐derived KIR2DS1‐positive clone is inactivated.

In that report [122], the proportion of donors with the KIR2DS1 gene was 33%. In the report by Yabe et al. [108], the KIR2DS1 gene‐positive rate was 38%. Since Yabe et al. focused only on recipients of HLA‐C2/C2, they did not analyze KIR2DS1‐positive donors because the number of cases was too small. In addition, it was reported that when the donors had KIR3DS1, although a decrease in the recurrence rate was not observed, the mortality rate decreased slightly [122]. Yabe et al. also analyzed leukemia patients and reported that transplantation from KIR3DS1 donors reduced the rate of acute GvHD [123]. It was also reported that the likelihood of acute GvHD increases when the donor is haplotype AA [124, 125]. The mechanisms underlying KIR/ HLA interactions remain unclear, but these reports may be a reference for donor selection.

#### **4.5. Immune checkpoint inhibitors**

#### *4.5.1. Checkpoint of NK cells*

Checkpoint inhibitors are an extremely promising approach among immunotherapies. Treatment with anti‐CTLA4 or anti‐PD‐1 antibody restored the T‐cell activity in cancer patients and resulted in tumor regression in several patients. A combination of both of the checkpoint inhibitors anti‐CTLA4 and anti‐PD‐1 could further enhance therapeutic benefits [126, 127]. It has been shown that NK cells from patients with multiple myeloma and renal cancer express PD‐1, the signal of which reduce the cytolytic activities of NK cells [128, 129]. Treatment using patient‐derived PD‐1+ NK cells with the anti‐PD‐1 antibody (pidilizumab, CT‐011) increased the NK cell‐mediated killing of autologous cancer cells *in vitro* [128]. The therapeutic benefit of activating PD‐1+ NK cells in cancer patients is currently not well understood, and the major therapeutic effect is certainly due to the re‐activation of exhausted T cells.

#### *4.5.2. Combination with a checkpoint inhibitor: expansion of the therapeutic spectrum*

A loss or down‐regulation of HLA class I antigens in tumor cells has been frequently observed in a variety of human malignancies, and this represents an important cancer‐immune escape mechanism [130–134]. Using a panel of monoclonal antibodies on tumor tissue sections, these loss or down‐regulation has been found in 60–90% of tumors [135–140]. Early studies using immunohistological analyses of different tumors showed a very low frequency of allelic loss. However, with the arrival of other techniques, such as studies of microsatellites to detect the loss of heterozygosity (LOH) on chromosome 6, it has been shown that LOH (haplotype loss) is the most frequent alteration of HLA class I expression [139, 141–144]. This alteration is caused by various defects in the HLA genomic region (i.e., the short arm of chromosome 6, 6p21), including chromosomal dysfunction, mitotic recombination, and genetic conversion.

The nature of the antigens that allow the immune system to distinguish cancer cells from non‐ cancer cells has long remained obscure. Recent technological innovations have made it possi‐ ble to dissect the immune response to patient‐specific neoantigens that arise as a consequence of tumor‐specific mutations, and emerging data suggest that the recognition of such neoanti‐ gens is a major factor in the activity of clinical immunotherapies. These observations indicate that the neoantigen load may form a biomarker in cancer immunotherapy and provide an incentive for the development of novel therapeutic approaches that selectively enhance T‐cell reactivity against this class of antigens.

If there is a neoantigen that can be a target of CTL and the patient has MHC on which the antigen is presented, and if the MHC is not lost from the tumor cells, treatments using CTL as an effector (e.g., checkpoint inhibitors) may be effective. NK cells that preferentially kill tumor cells whose expression of MHC has decreased by MHC non‐restriction are expected to have a synergistic effect with a checkpoint inhibitor.
